Antibodies binding to steap-1

ABSTRACT

The present invention generally relates to antibodies that bind to STEAP-1, including bispecific antigen binding molecules e.g. for activating T cells. In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and to methods of using them in the treatment of disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/587,606, filed Sep. 30, 2019, which is a continuation ofInternational Application No. PCT/EP2018/058043, filed Mar. 29, 2018,the entire contents of which are incorporated herein by reference, andwhich claims benefit under 35 U.S.C. § 119 to European PatentApplication No. 17164466.9, filed Apr. 3, 2017.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically via EFS-Web in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 10, 2021, isnamed “P34188-US-1_Sequence_Listing.txt” and is 68,947 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to antibodies that bind toSTEAP-1, including bispecific antigen binding molecules e.g. foractivating T cells. In addition, the present invention relates topolynucleotides encoding such antibodies, and vectors and host cellscomprising such polynucleotides. The invention further relates tomethods for producing the antibodies, and to methods of using them inthe treatment of disease.

BACKGROUND

STEAP-1 (six-transmembrane epithelial antigen of the prostate-1) is a339 amino acid cell surface protein which in normal tissues is expressedpredominantly in prostate cells. STEAP-1 protein expression ismaintained at high levels across various states of prostate cancer, andSTEAP-1 is also highly over-expressed in other human cancers such aslung and colon. The expression profile of STEAP-1 in normal and cancertissues suggested its potential use as a therapeutic target, e.g. forimmunotherapy. WO 2008/052187 reports anti-STEAP-1 antibodies andimmunoconjugates thereof. A STEAP-1/CD3 (scFv)₂ bispecific antibody isdescribed in WO 2014/165818. There exists a need for additional drugs totreat various cancers and metastases of cancers in the prostate, lungand colon. Particularly useful drugs for this purpose include antibodiesthat bind STEAP-1, in particular bispecific antibodies that bind STEAP-1on target cells and an activating T cell antigen such as CD3 on T-cells.The simultaneous binding of such an antibody to both of its targets willforce a temporary interaction between target cell and T cell, causingactivation of any cytotoxic T cell and subsequent lysis of the targetcell.

For therapeutic purposes, an important requirement that antibodies haveto fulfill is sufficient stability both in vitro (for storage of thedrug) an in vivo (after administration to the patient).

Modifications like asparagine deamidation, aspartate isomerization,succinimide formation, and tryptophane oxidation are typicaldegradations for recombinant antibodies and can affect both in vitrostability and in vivo biological functions.

The present invention provides novel antibodies, including bispecificantibodies, that bind STEAP-1 and are resistant to degradation by e.g.succinimide formation and thus show good stability. The (bispecific)antibodies provided further combine good efficacy and producibility withlow toxicity and favorable pharmacokinetic properties.

SUMMARY OF THE INVENTION

The present inventors have developed a novel antibody with unexpected,improved properties, that binds to STEAP-1. In particular, the antibodyis resistant to degradation e.g. by succinimide formation, and thusparticularly stable as required for therapeutic purposes. Furthermore,the inventors have developed bispecific antigen binding molecules thatbind to STEAP-1 and an activating T cell antigen, incorporating thenovel STEAP-1 antibody.

Thus, in a first aspect the present invention provides an antibody thatbinds to STEAP-1, wherein the antibody shows less than about 5%succinimide degradation after 4 weeks at pH 7.4, 37° C., and/or lessthan about 10% succinimide degradation after 4 weeks at pH 6.0, 40° C.,as determined by mass spectrometry.

In a further aspect the present invention provides an antibody thatbinds to STEAP-1, wherein the antibody comprises a heavy chain variableregion (VH) comprising a heavy chain complementary determining region(HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQID NO: 6, and a light chain variable region (VL) comprising a lightchain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, aLCDR 2 of SEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9. In one embodiment,the VH comprises an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to an amino acid sequence selected fromthe group of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the VLcomprises an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14.In one embodiment, the antibody is an IgG, particularly an IgG₁,antibody. In one embodiment, the antibody is a full-length antibody. Inanother embodiment, the antibody is an antibody fragment selected fromthe group of an Fv molecule, a scFv molecule, a Fab molecule, and aF(ab′)₂ molecule. In one embodiment, the antibody is a multispecificantibody.

The invention also provides a bispecific antigen binding molecule,comprising (a) a first antigen binding moiety that binds to a firstantigen, wherein the first antigen is STEAP-1 and the first antigenbinding moiety comprises a heavy chain variable region (VH) comprising aheavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 1, aHCDR 2 of SEQ ID NO: 2, and a HCDR 3 selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and a light chainvariable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 7, a LCDR 2 of SEQ ID NO: 8and a LCDR 3 of SEQ ID NO: 9, and (b) a second antigen binding moietywhich specifically binds to a second antigen. In one embodiment, the VHof the first antigen binding moiety comprises an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to anamino acid sequence selected from the group of SEQ ID NO: 11, SEQ ID NO:12 and SEQ ID NO: 13, and the VL of the first antigen binding moietycomprises an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14.In one embodiment, the second antigen is CD3, particularly CD3ε. In oneembodiment, the second antigen binding moiety comprises a VH comprisinga HCDR 1 of SEQ ID NO: 15, a HCDR 2 of SEQ ID NO: 16, and a HCDR 3 ofSEQ ID NO: 17, and a VL comprising a LCDR 1 of SEQ ID NO: 18, a LCDR 2of SEQ ID NO: 19 and a LCDR 3 of SEQ ID NO: 20. In one embodiment, theVH of the second antigen binding moiety comprises an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 21, and the VL of the second antigenbinding moiety comprises an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 22. In one embodiment, the first and/or the second antigenbinding moiety is a Fab molecule. In one embodiment, the second antigenbinding moiety is a Fab molecule wherein the variable domains VL and VHor the constant domains CL and CH1, particularly the variable domains VLand VH, of the Fab light chain and the Fab heavy chain are replaced byeach other. In one embodiment, the first antigen binding moiety is a Fabmolecule wherein in the constant domain the amino acid at position 124is substituted independently by lysine (K), arginine (R) or histidine(H) (numbering according to Kabat) and the amino acid at position 123 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat), and in the constant domain CH1 the aminoacid at position 147 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index) and theamino acid at position 213 is substituted independently by glutamic acid(E), or aspartic acid (D) (numbering according to Kabat EU index). Inone embodiment, the first and the second antigen binding moiety arefused to each other, optionally via a peptide linker. In one embodiment,the first and the second antigen binding moiety are each a Fab moleculeand either (i) the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety, or (ii) the first antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding moiety.In one embodiment, the bispecific antigen binding molecule comprises athird antigen binding moiety. In one embodiment, the third antigenmoiety is identical to the first antigen binding moiety. In oneembodiment, the bispecific antigen binding molecule comprises an Fcdomain composed of a first and a second subunit. In one embodiment, thefirst, the second and, where present, the third antigen binding moietyare each a Fab molecule; and either (i) the second antigen bindingmoiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moietyand the first antigen binding moiety is fused at the C-terminus of theFab heavy chain to the N-terminus of the first subunit of the Fc domain,or (ii) the first antigen binding moiety is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety and the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst subunit of the Fc domain; and the third antigen binding moiety,where present, is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. In one embodiment,the Fc domain is an IgG, particularly an IgG₁, Fc domain. In oneembodiment, the Fc domain is a human Fc domain. In one embodiment, anamino acid residue in the CH3 domain of the first subunit of the Fcdomain is replaced with an amino acid residue having a larger side chainvolume, thereby generating a protuberance within the CH3 domain of thefirst subunit which is positionable in a cavity within the CH3 domain ofthe second subunit, and an amino acid residue in the CH3 domain of thesecond subunit of the Fc domain is replaced with an amino acid residuehaving a smaller side chain volume, thereby generating a cavity withinthe CH3 domain of the second subunit within which the protuberancewithin the CH3 domain of the first subunit is positionable. In oneembodiment, the Fc domain comprises one or more amino acid substitutionthat reduces binding to an Fc receptor and/or effector function.

According to another aspect of the invention there is provided one ormore isolated polynucleotide(s) encoding an antibody or bispecificantigen binding molecule of the invention. The invention furtherprovides one or more expression vector(s) comprising the isolatedpolynucleotide(s) of the invention, and a host cell comprising theisolated polynucleotide(s) or the expression vector(s) of the invention.In some embodiments the host cell is a eukaryotic cell, particularly amammalian cell.

In another aspect is provided a method of producing an antibody thatbinds to STEAP-1, comprising the steps of a) culturing the host cell ofthe invention under conditions suitable for the expression of theantibody and b) recovering the antibody. The invention also encompassesan antibody that binds to STEAP-1 produced by the method of theinvention.

The invention further provides a pharmaceutical composition comprisingthe antibody or bispecific antigen binding molecule of the invention anda pharmaceutically acceptable carrier.

Also encompassed by the invention are methods of using the antibody,bispecific antigen binding molecule and pharmaceutical composition ofthe invention. In one aspect the invention provides an antibody,bispecific antigen binding molecule or pharmaceutical compositionaccording to the invention for use as a medicament. In one aspect isprovided an antibody, bispecific antigen binding molecule orpharmaceutical composition according to the invention for use in thetreatment of a disease. In a specific embodiment the disease is cancer.

Also provided is the use of an antibody or bispecific antigen bindingmolecule according to the invention in the manufacture of a medicamentfor the treatment of a disease; as well as a method of treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a composition comprising theantibody or bispecific antigen binding molecule according to theinvention in a pharmaceutically acceptable form. In a specificembodiment the disease is cancer. In any of the above embodiments theindividual preferably is a mammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1Z. Exemplary configurations of the bispecific antigenbinding molecules of the invention. (FIG. 1A, FIG. 1D) Illustration ofthe “1+1 CrossMab” molecule. (FIG. 1B, FIG. 1E) Illustration of the “2+1IgG Crossfab” molecule with alternative order of Crossfab and Fabcomponents (“inverted”). (FIG. 1C, FIG. 1F) Illustration of the “2+1 IgGCrossfab” molecule. (FIG. 1G, FIG. 1K) Illustration of the “1+1 IgGCrossfab” molecule with alternative order of Crossfab and Fab components(“inverted”). (FIG. 1H, FIG. 1L) Illustration of the “1+1 IgG Crossfab”molecule. (FIG. 1I, FIG. 1M) Illustration of the “2+1 IgG Crossfab”molecule with two CrossFabs. (FIG. 1J, FIG. 1N) Illustration of the “2+1IgG Crossfab” molecule with two CrossFabs and alternative order ofCrossfab and Fab components (“inverted”). (FIG. 1O, FIG. 1S)Illustration of the “Fab-Crossfab” molecule. (FIG. 1P, FIG. 1T)Illustration of the “Crossfab-Fab” molecule. (FIG. 1Q, FIG. 1U)Illustration of the “(Fab)₂-Crossfab” molecule. (FIG. 1R, FIG. 1V)Illustration of the “Crossfab-(Fab)₂” molecule. (FIG. 1W, FIG. 1Y)Illustration of the “Fab-(Crossfab)₂” molecule. (FIG. 1X, FIG. 1Z)Illustration of the “(Crossfab)₂-Fab” molecule. Black dot: optionalmodification in the Fc domain promoting heterodimerization. ++, −−:amino acids of opposite charges optionally introduced in the CH1 and CLdomains. Crossfab molecules are depicted as comprising an exchange of VHand VL regions, but may—in embodiments wherein no charge modificationsare introduced in CH1 and CL domains—alternatively comprise an exchangeof the CH1 and CL domains.

FIG. 2. Illustration of the T-cell bispecific (TCB) antibody moleculesprepared in the Examples. All tested TCB antibody molecules wereproduced as “2+1 IgG CrossFab, inverted” with charge modifications(VH/VL exchange in CD3 binder, charge modifications in STEAP1 binders,EE=147E, 213E: RK=123R, 124K).

FIG. 3A-FIG. 33. Sequence analysis of the variable domains ofvandortuzumab. (FIG. 3A) Prediction of hotspot positions in thesequence. (FIG. 3B) Annotation of the CDR regions and the predictedhotspots in the variable domain sequences of vandortuzumab.

FIG. 4A-FIG. 4B. T-cell mediated lysis of STEAP-1 expressing LnCAP cellsafter 24 h (FIG. 4A) or 48 h (FIG. 4B), induced by different STEAP-1 TCBantibody molecules (E:T=10:1, human PBMC effector cells). Depicted aretriplicates with SD.

FIG. 5A-FIG. 5C. Jurkat activation, as determined by luminescence, uponsimultaneous binding of different STEAP-1 TCB antibody molecules tohuman CD3 on Jurkat-NFAT reporter cells and human STEAP-1 on LnCAP (FIG.5A) or 22Rv1 cells (FIG. 5B). The STEAP-1-negative CHO-K1 cell lineserved as control (FIG. 5C). Depicted are triplicates with SD.

FIG. 6A-FIG. 6B. Binding of STEAP-1 TCB antibody molecules to humanSTEAP-1-expressing CHO-hSTEAP1 cells (FIG. 6A) and CD3-expressing JurkatNFAT cells (FIG. 6B).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are immunoglobulinsand derivatives. e.g. fragments, thereof.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises two antigenbinding sites, each of which is specific for a different antigenicdeterminant. In certain embodiments the bispecific antigen bindingmolecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. a second antigen bindingmoiety) to a target site, for example to a specific type of tumor cellbearing the antigenic determinant. In another embodiment an antigenbinding moiety is able to activate signaling through its target antigen,for example a T cell receptor complex antigen. Antigen binding moietiesinclude antibodies and fragments thereof as further defined herein.Particular antigen binding moieties include an antigen binding domain ofan antibody, comprising an antibody heavy chain variable region and anantibody light chain variable region. In certain embodiments, theantigen binding moieties may comprise antibody constant regions asfurther defined herein and known in the art. Useful heavy chain constantregions include any of the five isotypes: α, δ, ε, γ, or μ. Useful lightchain constant regions include any of the two isotypes: κ and λ.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope”, and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins referred to as antigens herein(e.g. STEAP-1, CD3) can be any native form of the proteins from anyvertebrate source, including mammals such as primates (e.g. humans),non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. In a particular embodiment theantigen is a human protein. Where reference is made to a specificprotein herein, the term encompasses the “full-length”, unprocessedprotein as well as any form of the protein that results from processingin the cell. The term also encompasses naturally occurring variants ofthe protein. e.g. splice variants or allelic variants. An exemplaryhuman protein useful as antigen is CD3, particularly the epsilon subunitof CD3 (see UniProt no. P07766 (version 185), NCBI RefSeq no.NP_000724.1, SEQ ID NO: 24 for the human sequence; or UniProt no. Q95LI5(version 69), NCBI GenBank no. BAB71849.1, SEQ ID NO: 25 for thecynomolgus [Macaca fascicularis] sequence), or STEAP-1(six-transmembrane epithelial antigen of prostate 1; see UniProt no.Q9UHE8 (version 137): NCBI RefSeq no. NP_036581.1, SEQ ID NO: 23 for thehuman sequence). In certain embodiments the antibody or bispecificantigen binding molecule of the invention binds to an epitope of CD3 orSTEAP-1 that is conserved among the CD3 or STEAP-1 antigens fromdifferent species. In particular embodiments, the antibody or bispecificantigen binding molecule of the invention binds to human STEAP-1.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding moiety to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance (SPR)technique (analyzed e.g. on a BIAcore instrument) (Liljeblad et al.,Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley,Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of bindingof an antigen binding moiety to an unrelated protein is less than about10% of the binding of the antigen binding moiety to the antigen asmeasured, e.g., by SPR. In certain embodiments, an antigen bindingmoiety that binds to the antigen, or an antigen binding moleculecomprising that antigen binding moiety, has a dissociation constant(K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ Mto 10⁻¹³ M).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., an antigen binding moiety and an antigen, or areceptor and its ligand). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (K_(D)), whichis the ratio of dissociation and association rate constants (k_(off) andk_(on), respectively). Thus, equivalent affinities may comprisedifferent rate constants, as long as the ratio of the rate constantsremains the same. Affinity can be measured by well established methodsknown in the art, including those described herein. A particular methodfor measuring affinity is Surface Plasmon Resonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity, the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

An “activating T cell antigen” as used herein refers to an antigenicdeterminant expressed on the surface of a T lymphocyte, particularly acytotoxic T lymphocyte, which is capable of inducing T cell activationupon interaction with an antigen binding molecule. Specifically,interaction of an antigen binding molecule with an activating T cellantigen may induce T cell activation by triggering the signaling cascadeof the T cell receptor complex. In a particular embodiment theactivating T cell antigen is CD3, particularly the epsilon subunit ofCD3 (see UniProt no. P07766 (version 144), NCBI RefSeq no. NP_000724.1,SEQ ID NO. 24 for the human sequence; or UniProt no. Q95LI5 (version49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 25 for the cynomolgus[Macaca fascicularis] sequence).

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. Suitable assays to measure T cell activation areknown in the art and described herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma. Ina particular embodiment, the target cell antigen is STEAP-1,particularly human STEAP-1.

As used herein, the terms “first”, “second” or “third” with respect toFab molecules etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of the bispecificantigen binding molecule unless explicitly so stated.

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein the variable domains or the constant domains of the Fabheavy and light chain are exchanged (i.e. replaced by each other), i.e.the crossover Fab molecule comprises a peptide chain composed of thelight chain variable domain VL and the heavy chain constant domain 1 CH1(VL-CH1, in N- to C-terminal direction), and a peptide chain composed ofthe heavy chain variable domain VH and the light chain constant domainCL (VH-CL, in N- to C-terminal direction). For clarity, in a crossoverFab molecule wherein the variable domains of the Fab light chain and theFab heavy chain are exchanged, the peptide chain comprising the heavychain constant domain 1 CH1 is referred to herein as the “heavy chain”of the (crossover) Fab molecule. Conversely, in a crossover Fab moleculewherein the constant domains of the Fab light chain and the Fab heavychain are exchanged, the peptide chain comprising the heavy chainvariable domain VH is referred to herein as the “heavy chain” of the(crossover) Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant domains (VH-CH1, in N- toC-terminal direction), and a light chain composed of the light chainvariable and constant domains (VL-CL, in N- to C-terminal direction).

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150.000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable domain (VH), also called a variable heavy domain or a heavychain variable region, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable domain (VL), also called avariable light domain or a light chain variable region, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprised in the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

An “isolated” antibody is one which has been separated from a componentof its natural environment. i.e. that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedantibody can be removed from its native or natural environment.

Recombinantly produced antibodies expressed in host cells are consideredisolated for the purpose of the invention, as are native or recombinantantibodies which have been separated, fractionated, or partially orsubstantially purified by any suitable technique. As such, theantibodies and bispecific antigen binding molecules of the presentinvention are isolated. In some embodiments, an antibody is purified togreater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC) methods. For review of methods for assessment of antibody purity,see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The terms “full length antibody.” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Pluckthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994): see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.: see e.g. U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable domain (VL) and an antibody heavy chainvariable domain (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity. As used herein in connection with variableregion sequences, “Kabat numbering” refers to the numbering system setforth by Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991).

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), referred to as “numberingaccording to Kabat” or “Kabat numbering” herein. Specifically the Kabatnumbering system (see pages 647-660 of Kabat, et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health. Bethesda, Md. (1991)) is used for thelight chain constant domain CL of kappa and lambda isotype and the KabatEU index numbering system (see pages 661-723) is used for the heavychain constant domains (CH1. Hinge, CH2 and CH3), which is hereinfurther clarified by referring to “numbering according to Kabat EUindex” in this case.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, Md. (1991));    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingorder in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. Such variable domains arereferred to herein as “humanized variable region”. A humanized antibodyoptionally may comprise at least a portion of an antibody constantregion derived from a human antibody. In some embodiments, some FRresidues in a humanized antibody are substituted with correspondingresidues from a non-human antibody (e.g., the antibody from which theHVR residues are derived), e.g., to restore or improve antibodyspecificity or affinity. A “humanized form” of an antibody. e.g. of anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. In certain embodiments, ahuman antibody is derived from a non-human transgenic mammal, forexample a mouse, a rat, or a rabbit. In certain embodiments, a humanantibody is derived from a hybridoma cell line. Antibodies or antibodyfragments isolated from human antibody libraries are also consideredhuman antibodies or human antibody fragments herein.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, antibodiesproduced by host cells may undergo post-translational cleavage of one ormore, particularly one or two, amino acids from the C-terminus of theheavy chain. Therefore an antibody produced by a host cell by expressionof a specific nucleic acid molecule encoding a full-length heavy chainmay include the full-length heavy chain, or it may include a cleavedvariant of the full-length heavy chain (also referred to herein as a“cleaved variant heavy chain”). This may be the case where the final twoC-terminal amino acids of the heavy chain are glycine (G446) and lysine(K447, numbering according to Kabat EU index). Therefore, the C-terminallysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447),of the Fc region may or may not be present. Amino acid sequences ofheavy chains including Fc domains (or a subunit of an Fc domain asdefined herein) are denoted herein without C-terminal glycine-lysinedipeptide if not indicated otherwise. In one embodiment of theinvention, a heavy chain including a subunit of an Fc domain asspecified herein, comprised in an antibody or bispecific antigen bindingmolecule according to the invention, comprises an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to EU indexof Kabat). In one embodiment of the invention, a heavy chain including asubunit of an Fc domain as specified herein, comprised in an antibody orbispecific antigen binding molecule according to the invention,comprises an additional C-terminal glycine residue (G446, numberingaccording to EU index of Kabat). Compositions of the invention, such asthe pharmaceutical compositions described herein, comprise a populationof antibodies or bispecific antigen binding molecules of the invention.The population of antibodies or bispecific antigen binding molecules maycomprise molecules having a full-length heavy chain and molecules havinga cleaved variant heavy chain. The population of antibodies orbispecific antigen binding molecules may consist of a mixture ofmolecules having a full-length heavy chain and molecules having acleaved variant heavy chain, wherein at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% of the antibodies or bispecificantigen binding molecules have a cleaved variant heavy chain. In oneembodiment of the invention a composition comprising a population ofantibodies or bispecific antigen binding molecules of the inventioncomprises an antibody or bispecific antigen binding molecule comprisinga heavy chain including a subunit of an Fc domain as specified hereinwith an additional C-terminal glycine-lysine dipeptide (G446 and K447,numbering according to EU index of Kabat). In one embodiment of theinvention a composition comprising a population of antibodies orbispecific antigen binding molecules of the invention comprises anantibody or bispecific antigen binding molecule comprising a heavy chainincluding a subunit of an Fc domain as specified herein with anadditional C-terminal glycine residue (G446, numbering according to EUindex of Kabat). In one embodiment of the invention such a compositioncomprises a population of antibodies or bispecific antigen bindingmolecules comprised of molecules comprising a heavy chain including asubunit of an Fc domain as specified herein; molecules comprising aheavy chain including a subunit of a Fc domain as specified herein withan additional C-terminal glycine residue (G446, numbering according toEU index of Kabat); and molecules comprising a heavy chain including asubunit of an Fc domain as specified herein with an additionalC-terminal glycine-lysine dipeptide (G446 and K447, numbering accordingto EU index of Kabat). Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991 (seealso above). A “subunit” of an Fc domain as used herein refers to one ofthe two polypeptides forming the dimeric Fc domain, i.e. a polypeptidecomprising C-terminal constant regions of an immunoglobulin heavy chain,capable of stable self-association. For example, a subunit of an IgG Fcdomain comprises an IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, omithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR)software or the FASTA program package. Those skilled in the art candetermine appropriate parameters for aligning sequences, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the ggsearch program of theFASTA package version 36.3.8c or later with a BLOSUM50 comparisonmatrix. The FASTA program package was authored by W. R. Pearson and D.J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”,PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequencecomparison” Meth. Enzymol. 266:227-258; and Pearson et. al. (1997)Genomics 46:24-36. Alternatively, a public server can be used to comparethe sequences, using the ggsearch (global protein:protein) program anddefault options (BLOSUM50; open: −10; ext: −2; Ktup=2) to ensure aglobal, rather than local, alignment is performed. Percent amino acididentity is given in the output alignment header.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

“Isolated polynucleotide (or nucleic acid) encoding [e.g. an antibody orbispecific antigen binding molecule of the invention]” refers to one ormore polynucleotide molecules encoding antibody heavy and light chains(or fragments thereof), including such polynucleotide molecule(s) in asingle vector or separate vectors, and such nucleic acid molecule(s)present at one or more locations in a host cell.

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette comprises polynucleotide sequencesthat encode antibodies or bispecific antigen binding molecules of theinvention or fragments thereof.

The term “vector” or “expression vector” refers to a DNA molecule thatis used to introduce and direct the expression of a specific gene towhich it is operably associated in a cell. The term includes the vectoras a self-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the cell,the ribonucleic acid molecule or protein that is encoded by the gene isproduced by the cellular transcription and/or translation machinery. Inone embodiment, the expression vector of the invention comprises anexpression cassette that comprises polynucleotide sequences that encodeantibodies or bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe antibodies or bispecific antigen binding molecules of the presentinvention. Host cells include cultured cells, e.g. mammalian culturedcells, such as HEK cells, CHO cells, BHK cells, NS0 cells. SP2/0 cells,YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells orhybridoma cells, yeast cells, insect cells, and plant cells, to nameonly a few, but also cells comprised within a transgenic animal,transgenic plant or cultured plant or animal tissue. An “activating Fcreceptor” is an Fc receptor that following engagement by an Fc domain ofan antibody elicits signaling events that stimulate the receptor-bearingcell to perform effector functions. Human activating Fc receptorsinclude FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI(CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g. PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe composition would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies orbispecific antigen binding molecules of the invention are used to delaydevelopment of a disease or to slow the progression of a disease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention provides antibodies and bispecific antigen bindingmolecules that bind STEAP-1, particularly human STEAP-1, and areresistant to resistant to degradation e.g. by succinimide formation, andthus particularly stable as required for therapeutic purposes. Inaddition, the molecules have also other favorable properties fortherapeutic application, e.g. with respect to efficacy and/or safety aswell as producibility.

STEAP-1 Antibody

Thus, in a first aspect the present invention provides an antibody thatbinds to STEAP-1, wherein the antibody shows less than about 5%succinimide degradation after 4 weeks at pH 7.4, 37° C., and/or lessthan about 10% succinimide degradation after 4 weeks at pH 6.0, 40° C.,as determined by mass spectrometry. In one embodiment, the antibodyshows less than about 3% succinimide degradation, particularly less thanabout 1% succinimide degradation, after 4 weeks at pH 7.4, 37° C., asdetermined by mass spectrometry. In one embodiment, the antibody showsless than about 7.5% succinimide degradation, particularly less thanabout 5% succinimide degradation, after 4 weeks at pH 6.0, 40° C., asdetermined by mass spectrometry.

In a further aspect the present invention provides an antibody thatbinds to STEAP-1, wherein the antibody comprises a heavy chain variableregion (VH) comprising a heavy chain complementary determining region(HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQID NO: 6, and a light chain variable region (VL) comprising a lightchain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, aLCDR 2 of SEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9.

In a particular embodiment, the antibody comprises a VH comprising aHCDR 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3 of SEQ IDNO: 6, and a VL comprising a LCDR 1 of SEQ ID NO: 7, a LCDR 2 of SEQ IDNO: 8 and a LCDR 3 of SEQ ID NO: 9.

In some embodiments, the antibody is a humanized antibody. In oneembodiment, the VH is a humanized VH and/or the VL is a humanized VL. Inone embodiment, the antibody comprises CDRs as in any of the aboveembodiments, and further comprises an acceptor human framework, e.g. ahuman immunoglobulin framework or a human consensus framework.

In one embodiment, the VH comprises an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 1000% identical to an amino acidsequence selected from the group of SEQ ID NO: 11, SEQ ID NO: 12 and SEQID NO: 13, and the VL comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 14.

In one embodiment, the antibody comprises a VH sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence selected from the group of SEQ ID NO: 11, SEQ ID NO: 12 and SEQID NO: 13, and a VL sequence that is at least about 95%, 96%, 97%, 98%,99% or 100% identical to the amino acid sequence of SEQ ID NO: 14.

In certain embodiments, a VH or VL sequence having at least 95%, 96%,97%, 98%, or 99% identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an antibody comprising that sequence retains the abilityto bind to STEAP-1. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in SEQ ID NO: 11,12 or 13 and/or a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 14. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the antibody comprises the VHsequence in SEQ ID NO: 11, 12 or 13 and/or the VL sequence in SEQ IDNO:14, including post-translational modifications of that sequence.

In one embodiment, the antibody comprises a VH comprising an amino acidsequence selected from the group of SEQ ID NO: 11, SEQ ID NO: 12 and SEQID NO: 13, and a VL comprising the amino acid sequence of SEQ ID NO: 14.

In one embodiment, the antibody comprises a VH sequence selected fromthe group of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO. 13, and the VLsequence of SEQ ID NO: 14.

In a particular embodiment, the antibody comprises a VH comprising theamino acid sequence of SEQ ID NO: 13 and a VL comprising the amino acidsequence of SEQ ID NO: 14.

In a particular embodiment, the antibody comprises the VH sequence ofSEQ ID NO: 13 and the VL sequence of SEQ ID NO: 14.

In one embodiment, the antibody comprises a human constant region. Inone embodiment, the antibody is an immunoglobulin molecule comprising ahuman constant region, particularly an IgG class immunoglobulin moleculecomprising a human CH1, CH2, CH3 and/or CL domain.

Exemplary sequences of human constant domains are given in SEQ ID NOs 39and 40 (human kappa and lambda CL domains, respectively) and SEQ ID NO:41 (human IgG1 heavy chain constant domains CH1-CH2-CH3). In someembodiments, the antibody comprises a light chain constant regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39or SEQ ID NO: 40, particularly the amino acid sequence of SEQ ID NO: 39.In some embodiments, the antibody comprises a heavy chain constantregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 41. Particularly, the heavy chain constant region may compriseamino acid mutations in the Fc domain as described herein.

In one embodiment, the antibody is a monoclonal antibody.

In one embodiment, the antibody is an IgG, particularly an IgG₁,antibody. In one embodiment, the antibody is a full-length antibody.

In one embodiment, the antibody comprises an Fc domain, particularly anIgG Fc domain, more particularly an IgG1 Fc domain. In one embodimentthe Fc domain is a human Fc domain. The Fc domain of the antibody mayincorporate any of the features, singly or in combination, describedherein in relation to the Fc domain of the bispecific antigen bindingmolecule of the invention.

In another embodiment, the antibody is an antibody fragment selectedfrom the group of an Fv molecule, a scFv molecule, a Fab molecule, and aF(ab′)₂ molecule; particularly a Fab molecule.

In another embodiment, the antibody fragment is a diabody, a triabody ora tetrabody.

In a further aspect, the antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in the sections below.

Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the oligosaccharide attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having anon-fucosylated oligosaccharide, i.e. an oligosaccharide structure thatlacks fucose attached (directly or indirectly) to an Fc region. Suchnon-fucosylated oligosaccharide (also referred to as “afucosylated”oligosaccharide) particularly is an N-linked oligosaccharide which lacksa fucose residue attached to the first GlcNAc in the stem of thebiantennary oligosaccharide structure. In one embodiment, antibodyvariants are provided having an increased proportion of non-fucosylatedoligosaccharides in the Fc region as compared to a native or parentantibody. For example, the proportion of non-fucosylatedoligosaccharides may be at least about 20%, at least about 40%, at leastabout 60%, at least about 80%, or even about 100% (i.e. no fucosylatedoligosaccharides are present). The percentage of non-fucosylatedoligosaccharides is the (average) amount of oligosaccharides lackingfucose residues, relative to the sum of all oligosaccharides attached toAsn 297 (e. g. complex, hybrid and high mannose structures) as measuredby MALDI-TOF mass spectrometry, as described in WO 2006/082515, forexample. Asn297 refers to the asparagine residue located at aboutposition 297 in the Fc region (EU numbering of Fc region residues);however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such antibodies having anincreased proportion of non-fucosylated oligosaccharides in the Fcregion may have improved FcγRIIIa receptor binding and/or improvedeffector function, in particular improved ADCC function. See, e.g., US2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies with reducedfucosylation include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003/0157108; and WO 2004/056312, especially at Example 11), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng.87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688(2006); and WO2003/085107), or cells with reduced or abolished activityof a GDP-fucose synthesis or transporter protein (see, e.g.,US2004259150, US2005031613, US2004132140, US2004110282).

In a further embodiment, antibody variants are provided with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc region of the antibody is bisected by GlcNAc. Such antibodyvariants may have reduced fucosylation and/or improved ADCC function asdescribed above. Examples of such antibody variants are described, e.g.,in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al.,Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO2003/011878.

Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. Cysteine engineered antibodies may begenerated as described, e.g., in U.S. Pat. No. 7,521,541, 8,30,930,7,855,275, 9,000,130, or WO2016040856.

Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

Immunoconjugates

The invention also provides immunoconjugates comprising an anti-STEAP-1antibody as described herein conjugated (chemically bonded) to one ormore therapeutic agents such as cytotoxic agents, chemotherapeuticagents, drugs, growth inhibitory agents, toxins (e.g., protein toxins,enzymatically active toxins of bacterial, fungal, plant, or animalorigin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more of thetherapeutic agents mentioned above. The antibody is typically connectedto one or more of the therapeutic agents using linkers. An overview ofADC technology including examples of therapeutic agents and drugs andlinkers is set forth in Pharmacol Review 68:3-19 (2016).

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or 1123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992): U.S. Pat. No. 5,208,020) may be used.

The immunoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites, i.e., different epitopes on different antigens ordifferent epitopes on the same antigen. In certain embodiments, themultispecific antibody has three or more binding specificities. Incertain embodiments, one of the binding specificities is for STEAP-1 andthe other (two or more) specificity is for any other antigen. In certainembodiments, bispecific antibodies may bind to two (or more) differentepitopes of STEAP-1. Multispecific (e.g., bispecific) antibodies mayalso be used to localize cytotoxic agents or cells to cells whichexpress STEAP-1. Multispecific antibodies can be prepared as full lengthantibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see.e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26(1997)). Multi-specific antibodies may also be made by engineeringelectrostatic steering effects for making antibody Fc-heterodimericmolecules (see, e.g., WO 2009/089004); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992) and WO 2011/034605); using the common lightchain technology for circumventing the light chain mis-pairing problem(see, e.g., WO 98/50431); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. JImmunol. 147: 60 (1991).

Engineered antibodies with three or more antigen binding sites,including for example, “Octopus antibodies.” or DVD-Ig are also includedherein (see, e.g. WO 2001/77342 and WO 2008/024715). Other examples ofmultispecific antibodies with three or more antigen binding sites can befound in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO2010/145792,and WO 2013/026831. The bispecific antibody or antigen binding fragmentthereof also includes a “Dual Acting FAb” or “DAF” comprising an antigenbinding site that binds to STEAP-1 as well as another different antigen,or two different epitopes of STEAP-1 (see, e.g., US 2008/0069820 and WO2015/095539).

Multi-specific antibodies may also be provided in an asymmetric formwith a domain crossover in one or more binding arms of the same antigenspecificity, i.e. by exchanging the VH/VL domains (see e.g., WO2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, andKlein at al., MAbs 8 (2016) 1010-20). Asymmetrical Fab arms can also beengineered by introducing charged or non-charged amino acid mutationsinto domain interfaces to direct correct Fab pairing. See e.g., WO2016/172485.

Various further molecular formats for multispecific antibodies are knownin the art and are included herein (see e.g., Spiess et al., Mol Immunol67 (2015) 95-106).

A particular type of multispecific antibodies, also included herein, arebispecific antibodies designed to simultaneously bind to a surfaceantigen on a target cell, e.g., a tumor cell, and to an activating,invariant component of the T cell receptor (TCR) complex, such as CD3,for retargeting of T cells to kill target cells. Hence, in certainembodiments, an antibody provided herein is a multispecific antibody,particularly a bispecific antibody, wherein one of the bindingspecificities is for STEAP-1 and the other is for CD3.

Examples of bispecific antibody formats that may be useful for thispurpose include, but are not limited to, the so-called “BiTE”(bispecific T cell engager) molecules wherein two scFv molecules arefused by a flexible linker (see, e.g., WO2004/106381, WO2005/061547,WO2007/042261, and WO2008/119567, Nagorsen and Bäuerle, Exp Cell Res317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305(1996)) and derivatives thereof, such as tandem diabodies (“TandAb”;Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinityretargeting) molecules which are based on the diabody format but featurea C-terminal disulfide bridge for additional stabilization (Johnson etal., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which arewhole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., CancerTreat Rev 36, 458-467 (2010)). Particular T cell bispecific antibodyformats included herein are described in WO 2013/026833, WO2013/026839,WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

Bispecific Antigen Binding Molecules that Bind to STEAP-1 and a SecondAntigen

The invention also provides a bispecific antigen binding molecule, i.e.an antigen binding molecule that comprises at least two antigen bindingmoieties capable of specific binding to two distinct antigenicdeterminants (a first and a second antigen).

According to particular embodiments of the invention, the antigenbinding moieties comprised in the bispecific antigen binding moleculeare Fab molecules (i.e. antigen binding domains composed of a heavy anda light chain, each comprising a variable and a constant domain). In oneembodiment, the first and/or the second antigen binding moiety is a Fabmolecule. In one embodiment, said Fab molecule is human. In a particularembodiment, said Fab molecule is humanized. In yet another embodiment,said Fab molecule comprises human heavy and light chain constantdomains.

Preferably, at least one of the antigen binding moieties is a crossoverFab molecule. Such modification reduces mispairing of heavy and lightchains from different Fab molecules, thereby improving the yield andpurity of the bispecific antigen binding molecule of the invention inrecombinant production. In a particular crossover Fab molecule usefulfor the bispecific antigen binding molecule of the invention, thevariable domains of the Fab light chain and the Fab heavy chain (VL andVH, respectively) are exchanged. Even with this domain exchange,however, the preparation of the bispecific antigen binding molecule maycomprise certain side products due to a so-called Bence Jones-typeinteraction between mispaired heavy and light chains (see Schaefer etal, PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavyand light chains from different Fab molecules and thus increase thepurity and yield of the desired bispecific antigen binding molecule,charged amino acids with opposite charges may be introduced at specificamino acid positions in the CH1 and CL domains of either the Fabmolecule(s) binding to the first antigen (STEAP-1), or the Fab moleculebinding to the second antigen (e.g. an activating T cell antigen such asCD3), as further described herein. Charge modifications are made eitherin the conventional Fab molecule(s) comprised in the bispecific antigenbinding molecule (such as shown e.g. in FIG. 1A-FIG. 1C, FIG. 1G-FIG.1J), or in the VH/VL crossover Fab molecule(s) comprised in thebispecific antigen binding molecule (such as shown e.g. in FIG. 1D-FIG.1F, FIG. 1K-FIG. 1N) (but not in both). In particular embodiments, thecharge modifications are made in the conventional Fab molecule(s)comprised in the bispecific antigen binding molecule (which inparticular embodiments bind(s) to the first antigen, i.e. STEAP-1).

In a particular embodiment according to the invention, the bispecificantigen binding molecule is capable of simultaneous binding to the firstantigen (i.e. STEAP-1), and the second antigen (e.g. an activating Tcell antigen, particularly CD3). In one embodiment, the bispecificantigen binding molecule is capable of crosslinking a T cell and atarget cell by simultaneous binding STEAP-1 and an activating T cellantigen. In an even more particular embodiment, such simultaneousbinding results in lysis of the target cell, particularly a STEAP-1expressing tumor cell. In one embodiment, such simultaneous bindingresults in activation of the T cell. In other embodiments, suchsimultaneous binding results in a cellular response of a T lymphocyte,particularly a cytotoxic T lymphocyte, selected from the group of:proliferation, differentiation, cytokine secretion, cytotoxic effectormolecule release, cytotoxic activity, and expression of activationmarkers. In one embodiment, binding of the bispecific antigen bindingmolecule to the activating T cell antigen, particularly CD3, withoutsimultaneous binding to STEAP-1 does not result in T cell activation.

In one embodiment, the bispecific antigen binding molecule is capable ofre-directing cytotoxic activity of a T cell to a target cell. In aparticular embodiment, said re-direction is independent of MHC-mediatedpeptide antigen presentation by the target cell and and/or specificityof the T cell. Particularly, a T cell according to any of theembodiments of the invention is a cytotoxic T cell. In some embodimentsthe T cell is a CD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

First Antigen Binding Moiety

The bispecific antigen binding molecule of the invention comprises atleast one antigen binding moiety, particularly a Fab molecule, thatbinds to STEAP-1 (first antigen). In certain embodiments, the bispecificantigen binding molecule comprises two antigen binding moieties,particularly Fab molecules, which bind to STEAP-1. In a particular suchembodiment, each of these antigen binding moieties binds to the sameantigenic determinant. In an even more particular embodiment, all ofthese antigen binding moieties are identical, i.e. they comprise thesame amino acid sequences including the same amino acid substitutions inthe CH1 and CL domain as described herein (if any). In one embodiment,the bispecific antigen binding molecule comprises not more than twoantigen binding moieties, particularly Fab molecules, which bind toSTEAP-1.

In particular embodiments, the antigen binding moiety(ies) which bind toSTEAP-1 is/are a conventional Fab molecule. In such embodiments, theantigen binding moiety(ies) that binds to a second antigen is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL or the constant domains CH1 and CL of theFab heavy and light chains are exchanged/replaced by each other.

In alternative embodiments, the antigen binding moiety(ies) which bindto STEAP-1 is/are a crossover Fab molecule as described herein, i.e. aFab molecule wherein the variable domains VH and VL or the constantdomains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other. In such embodiments, the antigenbinding moiety(ies) that binds a second antigen is a conventional Fabmolecule.

The STEAP-1 binding moiety is able to direct the bispecific antigenbinding molecule to a target site, for example to a specific type oftumor cell that expresses STEAP-1.

The first antigen binding moiety of the bispecific antigen bindingmolecule may incorporate any of the features, singly or in combination,described herein in relation to the antibody that binds STEAP-1, unlessscientifically clearly unreasonable or impossible.

Thus, in one aspect, the invention provides a bispecific antigen bindingmolecule, comprising (a) a first antigen binding moiety that binds to afirst antigen, wherein the first antigen is STEAP-1 and the firstantigen binding moiety comprises a heavy chain variable region (VH)comprising a heavy chain complementary determining region (HCDR) 1 ofSEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3 selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and alight chain variable region (VL) comprising a light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 7, a LCDR 2 ofSEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9, and (b) a second antigenbinding moiety that binds to a second antigen.

In a particular embodiment, the first antigen binding moiety comprises aVH comprising a HCDR 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and aHCDR 3 of SEQ ID NO: 6, and a VL comprising a LCDR 1 of SEQ ID NO: 7, aLCDR 2 of SEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9.

In some embodiments, the first antigen binding moiety is (derived from)a humanized antibody.

In one embodiment, the VH is a humanized VH and/or the VL is a humanizedVL. In one embodiment, the first antigen binding moiety comprises CDRsas in any of the above embodiments, and further comprises an acceptorhuman framework, e.g. a human immunoglobulin framework or a humanconsensus framework.

In one embodiment, the VH of the first antigen binding moiety comprisesan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to an amino acid sequence selected from the group of SEQID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the VL of the firstantigen binding moiety comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 14.

In one embodiment, the first antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group of SEQ IDNO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and a VL sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 14.

In one embodiment, the first antigen binding moiety comprises a VHcomprising an amino acid sequence selected from the group of SEQ ID NO:11, SEQ ID NO: 12 and SEQ ID NO: 13, and a VL comprising the amino acidsequence of SEQ ID NO: 14.

In one embodiment, the first antigen binding moiety comprises a VHsequence selected from the group of SEQ ID NO: 11, SEQ ID NO: 12 and SEQID NO: 13, and the VL sequence of SEQ ID NO: 14.

In a particular embodiment, the first antigen binding moiety comprises aVH comprising the amino acid sequence of SEQ ID NO: 13 and a VLcomprising the amino acid sequence of SEQ ID NO: 14.

In a particular embodiment, the first antigen binding moiety comprisesthe VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 14.

In one embodiment, the first antigen binding moiety comprises a humanconstant region. In one embodiment, the first antigen binding moiety isa Fab molecule comprising a human constant region, particularly a humanCH1 and/or CL domain. Exemplary sequences of human constant domains aregiven in SEQ ID NOs 39 and 40 (human kappa and lambda CL domains,respectively) and SEQ ID NO: 41 (human IgG₁ heavy chain constant domainsCH1-CH2-CH3). In some embodiments, the first antigen binding moietycomprises a light chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40,particularly the amino acid sequence of SEQ ID NO: 39. Particularly, thelight chain constant region may comprise amino acid mutations asdescribed herein under “charge modifications” and/or may comprisedeletion or substitutions of one or more (particularly two) N-terminalamino acids if in a crossover Fab molecule. In some embodiments, thefirst antigen binding moiety comprises a heavy chain constant regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the CH1 domain sequence comprised in theamino acid sequence of SEQ ID NO: 41. Particularly, the heavy chainconstant region (specifically CH1 domain) may comprise amino acidmutations as described herein under “charge modifications”.

Second Antigen Binding Moiety

The bispecific antigen binding molecule of the invention comprises atleast one antigen binding moiety, particularly a Fab molecule, thatbinds to a second antigen (different from STEAP-1).

In particular embodiments, the antigen binding moiety that binds thesecond antigen is a crossover Fab molecule as described herein, i.e. aFab molecule wherein the variable domains VH and VL or the constantdomains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other. In such embodiments, the antigenbinding moiety(ies) that binds to the first antigen (i.e. STEAP-1) ispreferably a conventional Fab molecule. In embodiments where there ismore than one antigen binding moiety, particularly Fab molecule, thatbinds to STEAP-1 comprised in the bispecific antigen binding molecule,the antigen binding moiety that binds to the second antigen preferablyis a crossover Fab molecule and the antigen binding moieties that bindto STEAP-1 are conventional Fab molecules.

In alternative embodiments, the antigen binding moiety that binds to thesecond antigen is a conventional Fab molecule. In such embodiments, theantigen binding moiety(ies) that binds to the first antigen (i.e.STEAP-1) is a crossover Fab molecule as described herein, i.e. a Fabmolecule wherein the variable domains VH and VL or the constant domainsCH1 and CL of the Fab heavy and light chains are exchanged/replaced byeach other. In embodiments where there is more than one antigen bindingmoiety, particularly Fab molecule, that binds to a second antigencomprised in the bispecific antigen binding molecule, the antigenbinding moiety that binds to STEAP-1 preferably is a crossover Fabmolecule and the antigen binding moieties that bind to the secondantigen are conventional Fab molecules.

In some embodiments, the second antigen is an activating T cell antigen(also referred to herein as an “activating T cell antigen bindingmoiety, or activating T cell antigen binding Fab molecule”).

In a particular embodiment, the bispecific antigen binding moleculecomprises not more than one antigen binding moiety capable of specificbinding to an activating T cell antigen. In one embodiment thebispecific antigen binding molecule provides monovalent binding to theactivating T cell antigen.

In particular embodiments, the second antigen is CD3, particularly humanCD3 (SEQ ID NO: 24) or cynomolgus CD3 (SEQ ID NO: 25), most particularlyhuman CD3. In one embodiment the second antigen binding moiety iscross-reactive for (i.e. specifically binds to) human and cynomolgusCD3. In some embodiments, the second antigen is the epsilon subunit ofCD3 (CD3 epsilon).

In one embodiment, the second antigen binding moiety comprises a HCDR 1of SEQ ID NO: 15, a HCDR 2 of SEQ ID NO: 16, a HCDR 3 of SEQ ID NO: 17,a LCDR 1 of SEQ ID NO: 18, a LCDR 2 of SEQ ID NO: 19 and a LCDR 3 of SEQID NO: 20.

In one embodiment, the second antigen binding moiety comprises a VHcomprising a HCDR 1 of SEQ ID NO: 15, a HCDR 2 of SEQ ID NO: 16, and aHCDR 3 of SEQ ID NO: 17, and a VL comprising a LCDR 1 of SEQ ID NO: 18,a LCDR 2 of SEQ ID NO: 19 and a LCDR 3 of SEQ ID NO: 20.

In some embodiments, the second antigen binding moiety is (derived from)a humanized antibody.

In one embodiment, the VH is a humanized VH and/or the VL is a humanizedVL. In one embodiment, the second antigen binding moiety comprises CDRsas in any of the above embodiments, and further comprises an acceptorhuman framework, e.g. a human immunoglobulin framework or a humanconsensus framework.

In one embodiment, the second antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 21. In oneembodiment, the second antigen binding moiety comprises a V L sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 22.

In one embodiment, the second antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 21, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 22.

In one embodiment, the VH of the second antigen binding moiety comprisesan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 21, and the VLof the second antigen binding moiety comprises an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 22.

In one embodiment, the second antigen binding moiety comprises a VHcomprising the amino acid sequence of SEQ ID NO: 21, and a VL comprisingthe amino acid sequence of SEQ ID NO: 22.

In one embodiment, the second antigen binding moiety comprises the VHsequence of SEQ ID NO: 21, and the VL sequence of SEQ ID NO: 22.

In one embodiment, the second antigen binding moiety comprises a humanconstant region. In one embodiment, the second antigen binding moiety isa Fab molecule comprising a human constant region, particularly a humanCH1 and/or CL domain. Exemplary sequences of human constant domains aregiven in SEQ ID NOs 39 and 40 (human kappa and lambda CL domains,respectively) and SEQ ID NO: 41 (human IgG₁ heavy chain constant domainsCH1-CH2-CH3). In some embodiments, the second antigen binding moietycomprises a light chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40,particularly the amino acid sequence of SEQ ID NO: 39. Particularly, thelight chain constant region may comprise amino acid mutations asdescribed herein under “charge modifications” and/or may comprisedeletion or substitutions of one or more (particularly two) N-terminalamino acids if in a crossover Fab molecule. In some embodiments, thesecond antigen binding moiety comprises a heavy chain constant regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the CH1 domain sequence comprised in theamino acid sequence of SEQ ID NO: 41. Particularly, the heavy chainconstant region (specifically CH1 domain) may comprise amino acidmutations as described herein under “charge modifications”.

In some embodiments, the second antigen binding moiety is a Fab moleculewherein the variable domains VL and VH or the constant domains CL andCH1, particularly the variable domains VL and VH, of the Fab light chainand the Fab heavy chain are replaced by each other (i.e. according tosuch embodiment, the second antigen binding moiety is a crossover Fabmolecule wherein the variable or constant domains of the Fab light chainand the Fab heavy chain are exchanged). In one such embodiment, thefirst (and the third, if any) antigen binding moiety is a conventionalFab molecule.

In one embodiment, not more than one antigen binding moiety that bindsto the second antigen (e.g. an activating T cell antigen such as CD3) ispresent in the bispecific antigen binding molecule (i.e. the bispecificantigen binding molecule provides monovalent binding to the secondantigen).

Charge Modifications

The bispecific antigen binding molecules of the invention may compriseamino acid substitutions in Fab molecules comprised therein which areparticularly efficient in reducing mispairing of light chains withnon-matching heavy chains (Bence-Jones-type side products), which canoccur in the production of Fab-based bi-/multispecific antigen bindingmolecules with a VH/VL exchange in one (or more, in case of moleculescomprising more than two antigen-binding Fab molecules) of their bindingarms (see also PCT publication no. WO 2015/150447, particularly theexamples therein, incorporated herein by reference in its entirety). Theratio of a desired bispecific antigen binding molecule compared toundesired side products, in particular Bence Jones-type side productsoccurring in bispecific antigen binding molecules with a VH/VL domainexchange in one of their binding arms, can be improved by theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH1 and CL domains (sometimes referred toherein as “charge modifications”).

Accordingly, in some embodiments wherein the first and the secondantigen binding moiety of the bispecific antigen binding molecule areboth Fab molecules, and in one of the antigen binding moieties(particularly the second antigen binding moiety) the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other,

i) in the constant domain CL of the first antigen binding moiety theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the first antigen binding moiety the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index); or

ii) in the constant domain CL of the second antigen binding moiety theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the second antigen binding moiety the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index).

The bispecific antigen binding molecule does not comprise bothmodifications mentioned under i) and ii). The constant domains CL andCH1 of the antigen binding moiety having the VH/VL exchange are notreplaced by each other (i.e. remain unexchanged).

In a more specific embodiment,

i) in the constant domain CL of the first antigen binding moiety theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the first antigen binding moiety the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index); or

ii) in the constant domain CL of the second antigen binding moiety theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In one such embodiment, in the constant domain CL of the first antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 or the amino acid atposition 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In a particular embodiment, in the constant domain CL of the firstantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the firstantigen binding moiety the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) (numbering according to Kabat), and inthe constant domain CH1 of the first antigen binding moiety the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an even more particular embodiment, in the constant domain CL of thefirst antigen binding moiety the amino acid at position 124 issubstituted by lysine (K) (numbering according to Kabat) and the aminoacid at position 123 is substituted by arginine (R) (numbering accordingto Kabat), and in the constant domain CH1 of the first antigen bindingmoiety the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index).

In particular embodiments, if amino acid substitutions according to theabove embodiments are made in the constant domain CL and the constantdomain CH1 of the first antigen binding moiety, the constant domain CLof the first antigen binding moiety is of kappa isotype.

Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second antigen binding moiety instead of in theconstant domain CL and the constant domain CH1 of the first antigenbinding moiety. In particular such embodiments, the constant domain CLof the second antigen binding moiety is of kappa isotype.

Accordingly, in one embodiment, in the constant domain CL of the secondantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 or the amino acidat position 213 is substituted independently by glutamic acid (E), oraspartic acid (D)(numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In still another embodiment, in the constant domain CL of the secondantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In one embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In another embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by arginine (R) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In a particular embodiment, the bispecific antigen binding molecule ofthe invention comprises

(a) a first antigen binding moiety that binds to a first antigen,wherein the first antigen is STEAP-1 and the first antigen bindingmoiety is a Fab molecule comprising a heavy chain variable region (VH)comprising a heavy chain complementary determining region (HCDR) 1 ofSEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3 selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and alight chain variable region (VL) comprising a light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 7, a LCDR 2 ofSEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9, and

(b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen binding moiety is a Fab molecule wherein thevariable domains VL and VH of the Fab light chain and the Fab heavychain are replaced by each other;

wherein in the constant domain CL of the first antigen binding moietythe amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) (in aparticular embodiment independently by lysine (K) or arginine (R)) andthe amino acid at position 123 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) (in aparticular embodiment independently by lysine (K) or arginine (R)), andin the constant domain CH1 of the first antigen binding moiety the aminoacid at position 147 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index) and theamino acid at position 213 is substituted independently by glutamic acid(E), or aspartic acid (D) (numbering according to Kabat EU index).

Bispecific Antigen Binding Molecule Formats

The components of the bispecific antigen binding molecule according tothe present invention can be fused to each other in a variety ofconfigurations. Exemplary configurations are depicted in FIG. 1A-FIG.1Z.

In particular embodiments, the antigen binding moieties comprised in thebispecific antigen binding molecule are Fab molecules. In suchembodiments, the first, second, third etc. antigen binding moiety may bereferred to herein as first, second, third etc. Fab molecule,respectively.

In one embodiment, the first and the second antigen binding moiety ofthe bispecific antigen binding molecule are fused to each other,optionally via a peptide linker. In particular embodiments, the firstand the second antigen binding moiety are each a Fab molecule. In onesuch embodiment, the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety. In another such embodiment,the first antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the secondantigen binding moiety. In embodiments wherein either (i) the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety or (ii) the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second antigen binding moiety, additionally the Fab lightchain of the first antigen binding moiety and the Fab light chain of thesecond antigen binding moiety may be fused to each other, optionally viaa peptide linker.

A bispecific antigen binding molecule with a single antigen bindingmoiety (such as a Fab molecule) capable of specific binding to a targetcell antigen such as STEAP-1 (for example as shown in FIG. 1A, FIG. 1D,FIG. 1G, FIG. 1H, FIG. 1K, FIG. 1L) is useful, particularly in caseswhere internalization of the target cell antigen is to be expectedfollowing binding of a high affinity antigen binding moiety. In suchcases, the presence of more than one antigen binding moiety specific forthe target cell antigen may enhance internalization of the target cellantigen, thereby reducing its availability.

In other cases, however, it will be advantageous to have a bispecificantigen binding molecule comprising two or more antigen binding moieties(such as Fab molecules) specific for a target cell antigen (see examplesshown in FIG. 1B, FIG. 1C, FIG. 1E, FIG. 1F, FIG. 1I, FIG. 1J, FIG. 1Mor FIG. 1N), for example to optimize targeting to the target site or toallow crosslinking of target cell antigens.

Accordingly, in particular embodiments, the bispecific antigen bindingmolecule according to the present invention comprises a third antigenbinding moiety.

In one embodiment, the third antigen binding moiety binds to the firstantigen, i.e. STEAP-1. In one embodiment, the third antigen bindingmoiety is a Fab molecule.

In one embodiment, the third antigen moiety is identical to the firstantigen binding moiety.

The third antigen binding moiety of the bispecific antigen bindingmolecule may incorporate any of the features, singly or in combination,described herein in relation to the first antigen binding moiety and/orthe antibody that binds STEAP-1, unless scientifically clearlyunreasonable or impossible.

In one embodiment, the third antigen binding moiety comprises a heavychain variable region (VH) comprising a heavy chain complementarydetermining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2,and a HCDR 3 selected from the group consisting of SEQ ID NO: 4, SEQ IDNO: 5 and SEQ ID NO: 6, and a light chain variable region (VL)comprising a light chain complementarity determining region (LCDR) 1 ofSEQ ID NO: 7, a LCDR 2 of SEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9.

In a particular embodiment, the third antigen binding moiety comprises aVH comprising a HCDR 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and aHCDR 3 of SEQ ID NO: 6, and a VL comprising a LCDR 1 of SEQ ID NO: 7, aLCDR 2 of SEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9.

In some embodiments, the third antigen binding moiety is (derived from)a humanized antibody.

In one embodiment, the VH is a humanized VH and/or the VL is a humanizedVL. In one embodiment, the third antigen binding moiety comprises CDRsas in any of the above embodiments, and further comprises an acceptorhuman framework, e.g. a human immunoglobulin framework or a humanconsensus framework.

In one embodiment, the VH of the third antigen binding moiety comprisesan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to an amino acid sequence selected from the group of SEQID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the VL of the thirdantigen binding moiety comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 14.

In one embodiment, the third antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group of SEQ IDNO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and a VL sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 14.

In one embodiment, the third antigen binding moiety comprises a VHcomprising an amino acid sequence selected from the group of SEQ ID NO:11, SEQ ID NO: 12 and SEQ ID NO: 13, and a VL comprising the amino acidsequence of SEQ ID NO: 14.

In one embodiment, the third antigen binding moiety comprises a VHsequence selected from the group of SEQ ID NO: 11, SEQ ID NO: 12 and SEQID NO: 13, and the VL sequence of SEQ ID NO: 14.

In a particular embodiment, the third antigen binding moiety comprises aVH comprising the amino acid sequence of SEQ ID NO: 13 and a VLcomprising the amino acid sequence of SEQ ID NO: 14.

In a particular embodiment, the third antigen binding moiety comprisesthe VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 14.

In one embodiment, the third antigen binding moiety comprises a humanconstant region. In one embodiment, the third antigen binding moiety isa Fab molecule comprising a human constant region, particularly a humanCH1 and/or CL domain. Exemplary sequences of human constant domains aregiven in SEQ ID NOs 39 and 40 (human kappa and lambda CL domains,respectively) and SEQ ID NO: 41 (human IgG₁ heavy chain constant domainsCH1-CH2-CH3). In some embodiments, the third antigen binding moietycomprises a light chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40,particularly the amino acid sequence of SEQ ID NO: 39. Particularly, thelight chain constant region may comprise amino acid mutations asdescribed herein under “charge modifications” and/or may comprisedeletion or substitutions of one or more (particularly two) N-terminalamino acids if in a crossover Fab molecule. In some embodiments, thethird antigen binding moiety comprises a heavy chain constant regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the CH1 domain sequence comprised in theamino acid sequence of SEQ ID NO: 41. Particularly, the heavy chainconstant region (specifically CH1 domain) may comprise amino acidmutations as described herein under “charge modifications”.

In particular embodiments, the third and the first antigen bindingmoiety are each a Fab molecule and the third antigen binding moiety isidentical to the first antigen binding moiety. Thus, in theseembodiments the first and the third antigen binding moiety comprise thesame heavy and light chain amino acid sequences and have the samearrangement of domains (i.e. conventional or crossover)). Furthermore,in these embodiments, the third antigen binding moiety comprises thesame amino acid substitutions, if any, as the first antigen bindingmoiety. For example, the amino acid substitutions described herein as“charge modifications” will be made in the constant domain CL and theconstant domain CH1 of each of the first antigen binding moiety and thethird antigen binding moiety. Alternatively, said amino acidsubstitutions may be made in the constant domain CL and the constantdomain CH1 of the second antigen binding moiety (which in particularembodiments is also a Fab molecule), but not in the constant domain CLand the constant domain CH1 of the first antigen binding moiety and thethird antigen binding moiety.

Like the first antigen binding moiety, the third antigen binding moietyparticularly is a conventional Fab molecule. Embodiments wherein thefirst and the third antigen binding moieties are crossover Fab molecules(and the second antigen binding moiety is a conventional Fab molecule)are, however, also contemplated. Thus, in particular embodiments, thefirst and the third antigen binding moieties are each a conventional Fabmolecule, and the second antigen binding moiety is a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL or the constant domains CL and CH1 of the Fab heavyand light chains are exchanged/replaced by each other. In otherembodiments, the first and the third antigen binding moieties are each acrossover Fab molecule and the second antigen binding moiety is aconventional Fab molecule.

If a third antigen binding moiety is present, in a particular embodimentthe first and the third antigen moiety bind to STEAP-1, and the secondantigen binding moiety binds to a second antigen, particularly anactivating T cell antigen, more particularly CD3, most particularly CD3epsilon.

In particular embodiments, the bispecific antigen binding moleculecomprises an Fc domain composed of a first and a second subunit. Thefirst and the second subunit of the Fc domain are capable of stableassociation.

The bispecific antigen binding molecule according to the invention canhave different configurations, i.e. the first, second (and optionallythird) antigen binding moiety may be fused to each other and to the Fcdomain in different ways. The components may be fused to each otherdirectly or, preferably, via one or more suitable peptide linkers. Wherefusion of a Fab molecule is to the N-terminus of a subunit of the Fcdomain, it is typically via an immunoglobulin hinge region.

In some embodiments, the first and the second antigen binding moiety areeach a Fab molecule and the second antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orthe second subunit of the Fc domain. In such embodiments, the firstantigen binding moiety may be fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding moiety or to the N-terminus of the other one of the subunits ofthe Fc domain. In particular such embodiments, said first antigenbinding moiety is a conventional Fab molecule, and the second antigenbinding moiety is a crossover Fab molecule as described herein, i.e. aFab molecule wherein the variable domains VH and VL or the constantdomains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other.

In other such embodiments, said first Fab molecule is a crossover Fabmolecule and the second Fab molecule is a conventional Fab molecule.

In one embodiment, the first and the second antigen binding moiety areeach a Fab molecule, the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain, and the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety. In a specificembodiment, the bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the second Fabmolecule, and the second Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Such a configuration is schematically depicted in FIG. 1Gand FIG. 1K (with the second antigen binding domain in these examplesbeing a VH/VL crossover Fab molecule). Optionally, the Fab light chainof the first Fab molecule and the Fab light chain of the second Fabmolecule may additionally be fused to each other.

In another embodiment, the first and the second antigen binding moietyare each a Fab molecule and the first and the second antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain. In a specificembodiment, the bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first and the second Fab molecule are each fused at theC-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain. Such a configuration is schematicallydepicted in FIG. 1A and FIG. 1D (in these examples with the secondantigen binding domain being a VH/VL crossover Fab molecule and thefirst antigen binding moiety being a conventional Fab molecule). Thefirst and the second Fab molecule may be fused to the Fc domain directlyor through a peptide linker. In a particular embodiment the first andthe second Fab molecule are each fused to the Fc domain through animmunoglobulin hinge region.

In a specific embodiment, the immunoglobulin hinge region is a humanIgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain.

In some embodiments, the first and the second antigen binding moiety areeach a Fab molecule and the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain. In such embodiments, the second antigenbinding moiety may be fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second antigen bindingmoiety or (as described above) to the N-terminus of the other one of thesubunits of the Fc domain. In particular such embodiments, said firstantigen binding moiety is a conventional Fab molecule, and the secondantigen binding moiety is a crossover Fab molecule as described herein,i.e. a Fab molecule wherein the variable domains VH and VL or theconstant domains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said firstFab molecule is a crossover Fab molecule and the second Fab molecule isa conventional Fab molecule.

In one embodiment, the first and the second antigen binding moiety areeach a Fab molecule, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain, and the second antigen binding moietyis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding moiety. In a specificembodiment, the bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the second Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the first Fabmolecule, and the first Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Such a configuration is schematically depicted in FIG. 1Hand FIG. 1L (in these examples with the second antigen binding domainbeing a VH/VL crossover Fab molecule and the first antigen bindingmoiety being a conventional Fab molecule). Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In some embodiments, a third antigen binding moiety, particularly athird Fab molecule, is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the first or second subunit of the Fc domain. Inparticular such embodiments, said first and third Fab molecules are eacha conventional Fab molecule, and the second Fab molecule is a crossoverFab molecule as described herein, i.e. a Fab molecule wherein thevariable domains VH and VL or the constant domains CL and CH1 of the Fabheavy and light chains are exchanged/replaced by each other. In othersuch embodiments, said first and third Fab molecules are each acrossover Fab molecule and the second Fab molecule is a conventional Fabmolecule.

In a particular such embodiment, the second and the third antigenbinding moiety are each fused at the C-terminus of the Fab heavy chainto the N-terminus of one of the subunits of the Fc domain, and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule. Ina specific embodiment, the bispecific antigen binding moleculeessentially consists of the first, the second and the third Fabmolecule, the Fc domain composed of a first and a second subunit, andoptionally one or more peptide linkers, wherein the first Fab moleculeis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the second Fab molecule, and the second Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. Such a configurationis schematically depicted in FIG. 1B and FIG. 1E (in these examples withthe second antigen binding moiety being a VH/VL crossover Fab molecule,and the first and the third antigen binding moiety being a conventionalFab molecule), and FIG. 1J and FIG. 1N (in these examples with thesecond antigen binding moiety being a conventional Fab molecule, and thefirst and the third antigen binding moiety being a VH/VL crossover Fabmolecule). The second and the third Fab molecule may be fused to the Fcdomain directly or through a peptide linker. In a particular embodimentthe second and the third Fab molecule are each fused to the Fc domainthrough an immunoglobulin hinge region. In a specific embodiment, theimmunoglobulin hinge region is a human IgG₁ hinge region, particularlywhere the Fc domain is an IgG₁ Fc domain. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In another such embodiment, the first and the third antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety. In a specific embodiment, the bispecific antigen bindingmolecule essentially consists of the first, the second and the third Fabmolecule, the Fc domain composed of a first and a second subunit, andoptionally one or more peptide linkers, wherein the second Fab moleculeis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first Fab molecule, and the first Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. Such a configurationis schematically depicted in FIG. 1C and FIG. 1F (in these examples withthe second antigen binding moiety being a VH/VL crossover Fab molecule,and the first and the third antigen binding moiety being a conventionalFab molecule) and in FIG. 1I and FIG. 1M (in these examples with thesecond antigen binding moiety being a conventional Fab molecule, and thefirst and the third antigen binding moiety being a VH/VL crossover Fabmolecule). The first and the third Fab molecule may be fused to the Fcdomain directly or through a peptide linker. In a particular embodimentthe first and the third Fab molecule are each fused to the Fc domainthrough an immunoglobulin hinge region. In a specific embodiment, theimmunoglobulin hinge region is a human IgG₁ hinge region, particularlywhere the Fc domain is an IgG₁ Fc domain. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In configurations of the bispecific antigen binding molecule wherein aFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of each of the subunits of the Fc domain through animmunoglobulin hinge regions, the two Fab molecules, the hinge regionsand the Fc domain essentially form an immunoglobulin molecule. In aparticular embodiment the immunoglobulin molecule is an IgG classimmunoglobulin. In an even more particular embodiment the immunoglobulinis an IgG₁ subclass immunoglobulin. In another embodiment theimmunoglobulin is an IgG4 subclass immunoglobulin. In a furtherparticular embodiment the immunoglobulin is a human immunoglobulin. Inother embodiments the immunoglobulin is a chimeric immunoglobulin or ahumanized immunoglobulin. In one embodiment, the immunoglobulincomprises a human constant region, particularly a human Fc region.

In some of the bispecific antigen binding molecule of the invention, theFab light chain of the first Fab molecule and the Fab light chain of thesecond Fab molecule are fused to each other, optionally via a peptidelinker. Depending on the configuration of the first and the second Fabmolecule, the Fab light chain of the first Fab molecule may be fused atits C-terminus to the N-terminus of the Fab light chain of the secondFab molecule, or the Fab light chain of the second Fab molecule may befused at its C-terminus to the N-terminus of the Fab light chain of thefirst Fab molecule. Fusion of the Fab light chains of the first and thesecond Fab molecule further reduces mispairing of unmatched Fab heavyand light chains, and also reduces the number of plasmids needed forexpression of some of the bispecific antigen binding molecules of theinvention.

The antigen binding moieties may be fused to the Fc domain or to eachother directly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n) peptide linkers. “n” is generally an integer from 1 to 10,typically from 2 to 4. In one embodiment said peptide linker has alength of at least 5 amino acids, in one embodiment a length of 5 to100, in a further embodiment of 10 to 50 amino acids. In one embodimentsaid peptide linker is (GxS)_(n) or (GxS)_(n)G_(m) with G=glycine,S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3,4 or 5 and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in afurther embodiment x=4 and n=2. In one embodiment said peptide linker is(G₄S)₂. A particularly suitable peptide linker for fusing the Fab lightchains of the first and the second Fab molecule to each other is (G₄S)₂.An exemplary peptide linker suitable for connecting the Fab heavy chainsof the first and the second Fab fragments comprises the sequence(D)-(G₄S)₂ (SEQ ID NOs 37 and 38). Another suitable such linkercomprises the sequence (G₄S)₄. Additionally, linkers may comprise (aportion of) an immunoglobulin hinge region Particularly where a Fabmolecule is fused to the N-terminus of an Fc domain subunit, it may befused via an immunoglobulin hinge region or a portion thereof, with orwithout an additional peptide linker.

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)), and apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab heavychain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₃₎-CL₍₁₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)), and apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab lightchain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (VL₍₂₎-CH1₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In some embodiments, the bispecific antigen binding molecule comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (i.e. the second Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainvariable region is replaced by a light chain variable region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe first Fab molecule, which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In other embodiments, thebispecific antigen binding molecule comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the second Fabmolecule which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with an Fcdomain subunit (VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the bispecific antigen binding moleculefurther comprises a crossover Fab light chain polypeptide of the secondFab molecule, wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎), and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Inothers of these embodiments the bispecific antigen binding moleculefurther comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab moleculewhich in turn shares a carboxy-terminal peptide bond with the Fab lightchain polypeptide of the first Fab molecule (VH₍₂₎CL₍₂₎-VL₍₁₎-CL₍₁₎), ora polypeptide wherein the Fab light chain polypeptide of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainvariable region of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VL₍₁₎CL₍₁₎VH₍₂₎CL₍₂₎), as appropriate.

The bispecific antigen binding molecule according to these embodimentsmay further comprise (i) an Fc domain subunit polypeptide(CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of athird Fab molecule shares a carboxy-terminal peptide bond with an Fcdomain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chainpolypeptide of a third Fab molecule (VL₍₃₎CL₍₃₎). In certain embodimentsthe polypeptides are covalently linked, e.g., by a disulfide bond.

In some embodiments, the bispecific antigen binding molecule comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (i.e. the second Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainconstant region is replaced by a light chain constant region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe first Fab molecule, which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎CH2-CH3(-CH4)).In other embodiments, the bispecific antigen binding molecule comprisesa polypeptide wherein the Fab heavy chain of the first Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain variableregion of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-VH₍₂₎CL₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the bispecific antigen binding moleculefurther comprises a crossover Fab light chain polypeptide of the secondFab molecule, wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎CH1₍₂₎), and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Inothers of these embodiments the bispecific antigen binding moleculefurther comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab moleculewhich in turn shares a carboxy-terminal peptide bond with the Fab lightchain polypeptide of the first Fab molecule (VL₍₂₎-CH1₍₂₎-VL₍₁₎-CL₍₁₎),or a polypeptide wherein the Fab light chain polypeptide of the firstFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain variable region of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The bispecific antigen binding molecule according to these embodimentsmay further comprise (i) an Fc domain subunit polypeptide(CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of athird Fab molecule shares a carboxy-terminal peptide bond with an Fcdomain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chainpolypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In certain embodiments, the bispecific antigen binding molecule does notcomprise an Fc domain. In particular such embodiments, said first and,if present third Fab molecules are each a conventional Fab molecule, andthe second Fab molecule is a crossover Fab molecule as described herein.i.e. a Fab molecule wherein the variable domains VH and VL or theconstant domains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said firstand, if present third Fab molecules are each a crossover Fab moleculeand the second Fab molecule is a conventional Fab molecule.

In one such embodiment, the bispecific antigen binding moleculeessentially consists of the first and the second antigen binding moiety,and optionally one or more peptide linkers, wherein the first and thesecond antigen binding moiety are both Fab molecules and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingmoiety. Such a configuration is schematically depicted in FIG. 1O andFIG. 1S (in these examples with the second antigen binding domain beinga VH/VL crossover Fab molecule and the first antigen binding moietybeing a conventional Fab molecule).

In another such embodiment, the bispecific antigen binding moleculeessentially consists of the first and the second antigen binding moiety,and optionally one or more peptide linkers, wherein the first and thesecond antigen binding moiety are both Fab molecules and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety. Such a configuration is schematically depicted in FIG. 1P andFIG. 1T (in these examples with the second antigen binding domain beinga VH/VL crossover Fab molecule and the first antigen binding moietybeing a conventional Fab molecule).

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the bispecific antigen binding molecule furthercomprises a third antigen binding moiety, particularly a third Fabmolecule, wherein said third Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule. In certain such embodiments, the bispecific antigenbinding molecule essentially consists of the first, the second and thethird Fab molecule, and optionally one or more peptide linkers, whereinthe first Fab molecule is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule, andthe third Fab molecule is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first Fab molecule. Sucha configuration is schematically depicted in FIG. 1Q and FIG. 1U (inthese examples with the second antigen binding domain being a VH/VLcrossover Fab molecule and the first and the antigen binding moiety eachbeing a conventional Fab molecule), or FIG. 1X and FIG. 1Z (in theseexamples with the second antigen binding domain being a conventional Fabmolecule and the first and the third antigen binding moiety each being aVH/VL crossover Fab molecule).

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the bispecific antigen binding molecule furthercomprises a third antigen binding moiety, particularly a third Fabmolecule, wherein said third Fab molecule is fused at the N-terminus ofthe Fab heavy chain to the C-terminus of the Fab heavy chain of thefirst Fab molecule. In certain such embodiments, the bispecific antigenbinding molecule essentially consists of the first, the second and thethird Fab molecule, and optionally one or more peptide linkers, whereinthe second Fab molecule is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the first Fabmolecule, and the third Fab molecule is fused at the N-terminus of theFab heavy chain to the C-terminus of the Fab heavy chain of the firstFab molecule. Such a configuration is schematically depicted in FIG. 1Rand FIG. 1V (in these examples with the second antigen binding domainbeing a VH/VL crossover Fab molecule and the first and the antigenbinding moiety each being a conventional Fab molecule), or FIG. 1W andFIG. 1Y (in these examples with the second antigen binding domain beinga conventional Fab molecule and the first and the third antigen bindingmoiety each being a VH/VL crossover Fab molecule).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofthe first Fab molecule shares a carboxy-terminal peptide bond with theFab light chain variable region of the second Fab molecule, which inturn shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the second Fab molecule (i.e. the second Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain variableregion is replaced by a light chain variable region)(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab heavychain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule(VL₍₂₎-CH1₍₂₎-VH₍₂₎-CH1₍₂₎). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab heavychain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule(VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab lightchain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (VL₍₂₎-CH1₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab heavychain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofa third Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain of the first Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab light chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region)(VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In some embodiments the bispecific antigen binding molecule furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofa third Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain of the first Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region)(VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the bispecific antigen binding molecule furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of athird Fab molecule (VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the bispecific antigen binding molecule furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule, which in tumshares a carboxy-terminal peptide bond with the Fab heavy chain of athird Fab molecule (VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the bispecific antigen binding molecule furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofthe second Fab molecule shares a carboxy-terminal peptide bond with theFab light chain variable region of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab light chain variable regionof a third Fab molecule, which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain constant region of a third Fab molecule(i.e. the third Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region) (VH₍₂₎-CH1₍₂₎-VL₍₁₎-CH1₍₁₎-VL₍₃₎-CH1₍₃₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab light chainconstant region of the first Fab molecule (VH₍₁₎-CL₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). Insome embodiments the bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab heavy chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fablight chain constant region of a third Fab molecule (VH₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofthe second Fab molecule shares a carboxy-terminal peptide bond with theFab heavy chain variable region of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof a third Fab molecule, which in turn shares a carboxy-terminal peptidebond with the Fab light chain constant region of a third Fab molecule(i.e. the third Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain constant region is replaced by a light chainconstant region) (VH₍₂₎-CH1₍₂₎-VH₍₁₎-CL₍₁₎-VH₍₃₎-CL₍₃₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). Insome embodiments the bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab light chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain constant region of a third Fab molecule (VL₍₃₎-CH1₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab light chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of a third Fabmolecule (i.e. the third Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab light chain variable region of the first Fab molecule,which in turn shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the first Fab molecule (i.e. the first Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainvariable region is replaced by a light chain variable region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe second Fab molecule (VL₍₃₎-CH1₍₃₎-VL₍₁₎CH1₍₁₎-VH₍₂₎-CH1₍₂₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab light chainconstant region of the first Fab molecule (VH₍₁₎-CL₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). Insome embodiments the bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab heavy chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fablight chain constant region of a third Fab molecule (VH₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of a third Fabmolecule (i.e. the third Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain variable region of the first Fab molecule,which in turn shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the first Fab molecule (i.e. the first Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainconstant region is replaced by a light chain constant region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe second Fab molecule (VH₍₃₎-CL₍₃₎-VH₍₁₎-CL₍₁₎-VH₍₂₎-CH1₍₁₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). Insome embodiments the bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab light chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain constant region of a third Fab molecule (VL₍₃₎-CH1₍₃₎).

In one embodiment, the invention provides a bispecific antigen bindingmolecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is STEAP-1 and the first antigen binding moiety is aFab molecule comprising a heavy chain variable region (VH) comprising aheavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 1, aHCDR 2 of SEQ ID NO: 2, and a HCDR 3 selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and a light chainvariable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 7, a LCDR 2 of SEQ ID NO: 8and a LCDR 3 of SEQ ID NO: 9;

b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other; c) an Fc domain composed of a firstand a second subunit:

wherein

(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) is fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c), or

(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) is fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In a particular embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is STEAP-1 and the first antigen binding moiety is aFab molecule comprising a heavy chain variable region (VH) comprising aheavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 1, aHCDR 2 of SEQ ID NO: 2, and a HCDR 3 selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and a light chainvariable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 7, a LCDR 2 of SEQ ID NO: 8and a LCDR 3 of SEQ ID NO: 9;

b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other:

c) a third antigen binding moiety that binds to the first antigen and isidentical to the first antigen binding moiety; and

d) an Fc domain composed of a first and a second subunit;

wherein

(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under c) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under d), or

(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) and the third antigen binding moietyunder c) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under d).

In another embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is STEAP-1 and the first antigen binding moiety is aFab molecule comprising a heavy chain variable region (VH) comprising aheavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 1, aHCDR 2 of SEQ ID NO: 2, and a HCDR 3 selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and a light chainvariable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 7, a LCDR 2 of SEQ ID NO: 8and a LCDR 3 of SEQ ID NO: 9;

b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other:

c) an Fc domain composed of a first and a second subunit;

wherein

(i) the first antigen binding moiety under a) and the second antigenbinding moiety under b) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In all of the different configurations of the bispecific antigen bindingmolecule according to the invention, the amino acid substitutionsdescribed herein, if present, may either be in the CH1 and CL domains ofthe first and (if present) the third antigen binding moiety/Fabmolecule, or in the CH1 and CL domains of the second antigen bindingmoiety/Fab molecule. Preferably, they are in the CH1 and CL domains ofthe first and (if present) the third antigen binding moiety/Fabmolecule. In accordance with the concept of the invention, if amino acidsubstitutions as described herein are made in the first (and, ifpresent, the third) antigen binding moiety/Fab molecule, no such aminoacid substitutions are made in the second antigen binding moiety/Fabmolecule. Conversely, if amino acid substitutions as described hereinare made in the second antigen binding moiety/Fab molecule, no suchamino acid substitutions are made in the first (and, if present, thethird) antigen binding moiety/Fab molecule. Amino acid substitutions areparticularly made in bispecific antigen binding molecules comprising aFab molecule wherein the variable domains VL and VH1 of the Fab lightchain and the Fab heavy chain are replaced by each other.

In particular embodiments of the bispecific antigen binding moleculeaccording to the invention, particularly wherein amino acidsubstitutions as described herein are made in the first (and, ifpresent, the third) antigen binding moiety/Fab molecule, the constantdomain CL of the first (and, if present, the third) Fab molecule is ofkappa isotype. In other embodiments of the bispecific antigen bindingmolecule according to the invention, particularly wherein amino acidsubstitutions as described herein are made in the second antigen bindingmoiety/Fab molecule, the constant domain CL of the second antigenbinding moiety/Fab molecule is of kappa isotype. In some embodiments,the constant domain CL of the first (and, if present, the third) antigenbinding moiety/Fab molecule and the constant domain CL of the secondantigen binding moiety/Fab molecule are of kappa isotype.

In one embodiment, the invention provides a bispecific antigen bindingmolecule comprising a) a first antigen binding moiety that binds to afirst antigen, wherein the first antigen is STEAP-1 and the firstantigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQID NO: 6, and a light chain variable region (VL) comprising a lightchain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, aLCDR 2 of SEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9;

b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other;

c) an Fc domain composed of a first and a second subunit;

wherein in the constant domain CL of the first antigen binding moietyunder a) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first antigen binding moiety under a) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); and

wherein

(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) is fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c), or

(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) is fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In a particular embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is STEAP-1 and the first antigen binding moiety is aFab molecule comprising a heavy chain variable region (VH) comprising aheavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 1, aHCDR 2 of SEQ ID NO: 2, and a HCDR 3 selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and a light chainvariable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 7, a LCDR 2 of SEQ ID NO: 8and a LCDR 3 of SEQ ID NO: 9:

b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other:

c) a third antigen binding moiety that binds to the first antigen and isidentical to the first antigen binding moiety; and

d) an Fc domain composed of a first and a second subunit:

wherein in the constant domain CL of the first antigen binding moietyunder a) and the third antigen binding moiety under c) the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by lysine (K) orarginine (R) (numbering according to Kabat) (most particularly byarginine (R)), and wherein in the constant domain CH1 of the firstantigen binding moiety under a) and the third antigen binding moietyunder c) the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index); and

wherein

(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under c) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under d), or

(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) and the third antigen binding moietyunder c) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under d).

In another embodiment, the invention provides a bispecific antigenbinding molecule comprising a) a first antigen binding moiety that bindsto a first antigen, wherein the first antigen is STEAP-1 and the firstantigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQID NO: 6, and a light chain variable region (VL) comprising a lightchain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, aLCDR 2 of SEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9,

b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other;

c) an Fc domain composed of a first and a second subunit;

wherein in the constant domain CL of the first antigen binding moietyunder a) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first antigen binding moiety under a) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); and

wherein the first antigen binding moiety under a) and the second antigenbinding moiety under b) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

According to any of the above embodiments, components of the bispecificantigen binding molecule (e.g. Fab molecules, Fc domain) may be fuseddirectly or through various linkers, particularly peptide linkerscomprising one or more amino acids, typically about 2-20 amino acids,that are described herein or are known in the art. Suitable,non-immunogenic peptide linkers include, for example, (G₄S)_(n),(SG₄)_(n), (G₄S)_(n) or G₄(SG₄)_(n) peptide linkers, wherein n isgenerally an integer from 1 to 10, typically from 2 to 4.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

a) a first and a third antigen binding moiety that binds to a firstantigen; wherein the first antigen is STEAP-1 and wherein the first andthe second antigen binding moiety are each a (conventional) Fab moleculecomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 13 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 14;

b) a second antigen binding moiety that binds to a second antigen;wherein the second antigen is CD3 and wherein the second antigen bindingmoiety is Fab molecule wherein the variable domains VL and VH of the Fablight chain and the Fab heavy chain are replaced by each other,comprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 21 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 22:

c) an Fc domain composed of a first and a second subunit;

wherein

in the constant domain CL of the first and the third antigen bindingmoiety under a) the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first and the third antigen binding moiety under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex);

and wherein further

the first antigen binding moiety under a) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under a) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c).

In one embodiment according to this aspect of the invention, in thefirst subunit of the Fc domain the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in the second subunit ofthe Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V) and optionally the threonine residue at position366 is replaced with a serine residue (T366S) and the leucine residue atposition 368 is replaced with an alanine residue (L368A) (numberingsaccording to Kabat EU index).

In a further embodiment according to this aspect of the invention, inthe first subunit of the Fc domain additionally the serine residue atposition 354 is replaced with a cysteine residue (S354C) or the glutamicacid residue at position 356 is replaced with a cysteine residue (E356C)(particularly the serine residue at position 354 is replaced with acysteine residue), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C) (numberings according to Kabat EU index).

In still a further embodiment according to this aspect of the invention,in each of the first and the second subunit of the Fc domain the leucineresidue at position 234 is replaced with an alanine residue (L234A), theleucine residue at position 235 is replaced with an alanine residue(L235A) and the proline residue at position 329 is replaced by a glycineresidue (P329G) (numbering according to Kabat EU index).

In still a further embodiment according to this aspect of the invention,the Fc domain is a human IgG₁ Fc domain.

In particular specific embodiment, the bispecific antigen bindingmolecule comprises a polypeptide comprising an amino acid sequence thatis at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQID NO: 32, a polypeptide comprising an amino acid sequence that is atleast 95%, 96%, 97%, 98%, or 99;% identical to the sequence of SEQ IDNO: 33, a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 34,and a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 35.In a further particular specific embodiment, the bispecific antigenbinding molecule comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 32, a polypeptide comprising the amino acidsequence of SEQ ID NO: 33, a polypeptide comprising the amino acidsequence of SEQ ID NO: 34 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 35.

In another specific embodiment, the bispecific antigen binding moleculecomprises a polypeptide comprising an amino acid sequence that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:28, a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 29, apolypeptide comprising an amino acid sequence that is at least 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 34, and apolypeptide comprising an amino acid sequence that is at least 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 35. In afurther specific embodiment, the bispecific antigen binding moleculecomprises a polypeptide comprising the amino acid sequence of SEQ ID NO:28, a polypeptide comprising the amino acid sequence of SEQ ID NO: 29, apolypeptide comprising the amino acid sequence of SEQ ID NO: 34 and apolypeptide comprising the amino acid sequence of SEQ ID NO: 35.

In still another specific embodiment, the bispecific antigen bindingmolecule comprises a polypeptide comprising an amino acid sequence thatis at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQID NO: 30, a polypeptide comprising an amino acid sequence that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:61, a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 34,and a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 35.In a further specific embodiment, the bispecific antigen bindingmolecule comprises a polypeptide comprising the amino acid sequence ofSEQ ID NO: 30, a polypeptide comprising the amino acid sequence of SEQID NO: 31, a polypeptide comprising the amino acid sequence of SEQ IDNO: 34 and a polypeptide comprising the amino acid sequence of SEQ IDNO: 35.

Fc Domain

In particular embodiments, the bispecific antigen binding molecule ofthe invention comprises an Fc domain composed of a first and a secondsubunit. It is understood, that the features of the Fc domain describedherein in relation to the bispecific antigen binding molecule canequally apply to an Fc domain comprised in an antibody of the invention.

The Fc domain of the bispecific antigen binding molecule consists of apair of polypeptide chains comprising heavy chain domains of animmunoglobulin molecule. For example, the Fc domain of an immunoglobulinG (IgG) molecule is a dimer, each subunit of which comprises the CH2 andCH3 IgG heavy chain constant domains. The two subunits of the Fc domainare capable of stable association with each other. In one embodiment,the bispecific antigen binding molecule of the invention comprises notmore than one Fc domain.

In one embodiment, the Fc domain of the bispecific antigen bindingmolecule is an IgG Fc domain.

In a particular embodiment, the Fc domain is an IgG₁ Fc domain. Inanother embodiment the Fc domain is an IgG₄ Fc domain. In a morespecific embodiment, the Fc domain is an IgG₄ Fc domain comprising anamino acid substitution at position S228 (Kabat EU index numbering),particularly the amino acid substitution S228P. This amino acidsubstitution reduces in vivo Fab arm exchange of IgG₄ antibodies (seeStubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).

In a further particular embodiment, the Fc domain is a human Fc domain.In an even more particular embodiment, the Fc domain is a human IgG₁ Fcdomain. An exemplary sequence of a human IgG₁ Fc region is given in SEQID NO: 36.

Fc Domain Modifications Promoting Heterodimerization

Bispecific antigen binding molecules according to the invention comprisedifferent antigen binding moieties, which may be fused to one or theother of the two subunits of the Fc domain, thus the two subunits of theFc domain are typically comprised in two non-identical polypeptidechains. Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of bispecific antigenbinding molecules in recombinant production, it will thus beadvantageous to introduce in the Fc domain of the bispecific antigenbinding molecule a modification promoting the association of the desiredpolypeptides.

Accordingly, in particular embodiments, the Fc domain of the bispecificantigen binding molecule according to the invention comprises amodification promoting the association of the first and the secondsubunit of the Fc domain. The site of most extensive protein-proteininteraction between the two subunits of a human IgG Fc domain is in theCH3 domain of the Fc domain. Thus, in one embodiment said modificationis in the CH3 domain of the Fc domain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011. WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004. WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954. WO 2013096291. Typically, inall such approaches the CH3 domain of the first subunit of the Fc domainand the CH3 domain of the second subunit of the Fc domain are bothengineered in a complementary manner so that each CH3 domain (or theheavy chain comprising it) can no longer homodimerize with itself but isforced to heterodimerize with the complementarily engineered other CH3domain (so that the first and second CH3 domain heterodimerize and nohomdimers between the two first or the two second CH3 domains areformed). These different approaches for improved heavy chainheterodimerization are contemplated as different alternatives incombination with the heavy-light chain modifications (e.g. VH and VLexchange/replacement in one binding arm and the introduction ofsubstitutions of charged amino acids with opposite charges in the CH1/CLinterface) in the bispecific antigen binding molecule which reduceheavy/light chain mispairing and Bence Jones-type side products.

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter. J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain of the bispecific antigen binding molecule anamino acid residue is replaced with an amino acid residue having alarger side chain volume, thereby generating a protuberance within theCH3 domain of the first subunit which is positionable in a cavity withinthe CH3 domain of the second subunit, and in the CH3 domain of thesecond subunit of the Fc domain an amino acid residue is replaced withan amino acid residue having a smaller side chain volume, therebygenerating a cavity within the CH3 domain of the second subunit withinwhich the protuberance within the CH3 domain of the first subunit ispositionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in (the CH3 domain of) the first subunit ofthe Fc domain (the “knobs” subunit) the threonine residue at position366 is replaced with a tryptophan residue (T366W), and in (the CH3domain of) the second subunit of the Fc domain (the “hole” subunit) thetyrosine residue at position 407 is replaced with a valine residue(Y407V). In one embodiment, in the second subunit of the Fc domainadditionally the threonine residue at position 366 is replaced with aserine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A) (numberings according to KabatEU index).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C) (particularly the serineresidue at position 354 is replaced with a cysteine residue), and in thesecond subunit of the Fc domain additionally the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C) (numberingsaccording to Kabat EU index). Introduction of these two cysteineresidues results in formation of a disulfide bridge between the twosubunits of the Fc domain, further stabilizing the dimer (Carter, JImmunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprisesthe amino acid substitutions S354C and T366W, and the second subunit ofthe Fc domain comprises the amino acid substitutions Y349C, T366S, L368Aand Y407V (numbering according to Kabat EU index). In a particularembodiment the antigen binding moiety that binds to the second antigen(e.g. an activating T cell antigen) is fused (optionally via the firstantigen binding moiety, which binds to STEAP-1, and/or a peptide linker)to the first subunit of the Fc domain (comprising the “knob”modification). Without wishing to be bound by theory, fusion of theantigen binding moiety that binds a second antigen, such as anactivating T cell antigen, to the knob-containing subunit of the Fcdomain will (further) minimize the generation of antigen bindingmolecules comprising two antigen binding moieties that bind to anactivating T cell antigen (steric clash of two knob-containingpolypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one embodiment, the heterodimerization approach described in EP1870459, is used alternatively. This approach is based on theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH3/CH3 domain interface between the twosubunits of the Fc domain. One preferred embodiment for the bispecificantigen binding molecule of the invention are amino acid mutationsR409D; K370E in one of the two CH3 domains (of the Fc domain) and aminoacid mutations D399K; E357K in the other one of the CH3 domains of theFc domain (numbering according to Kabat EU index).

In another embodiment, the bispecific antigen binding molecule of theinvention comprises amino acid mutation T366W in the CH3 domain of thefirst subunit of the Fc domain and amino acid mutations T366S, L368A,Y407V in the CH3 domain of the second subunit of the Fc domain, andadditionally amino acid mutations R409D; K370E in the CH3 domain of thefirst subunit of the Fc domain and amino acid mutations D399K; E357K inthe CH3 domain of the second subunit of the Fc domain (numberingsaccording to Kabat EU index).

In another embodiment, the bispecific antigen binding molecule of theinvention comprises amino acid mutations S354C, T366W in the CH3 domainof the first subunit of the Fc domain and amino acid mutations Y349C,T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fcdomain, or said bispecific antigen binding molecule comprises amino acidmutations Y349C, T366W in the CH3 domain of the first subunit of the Fcdomain and amino acid mutations S354C. T366S, L368A, Y407V in the CH3domains of the second subunit of the Fc domain and additionally aminoacid mutations R409D: K370E in the CH3 domain of the first subunit ofthe Fc domain and amino acid mutations D399K; E357K in the CH3 domain ofthe second subunit of the Fc domain (all numberings according to KabatEU index).

In one embodiment, the heterodimerization approach described in WO2013/157953 is used alternatively. In one embodiment, a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (numberings according to Kabat EU index). In afurther embodiment, the first CH3 domain comprises further amino acidmutation L351K. In a further embodiment, the second CH3 domain comprisesfurther an amino acid mutation selected from Y349E, Y349D and L368E(preferably L368E) (numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2012/058768 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutations L351Y, Y407A and a second CH3 domaincomprises amino acid mutations T366A, K409F. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, S400, F405, N390, or K392, e.g. selected from a) T411 N,T411R, T411Q, T411K, T411 D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S,F405V or F405W, e) N390R. N390K or N390D, f) K392V. K392M, K392R, K392L,K392F or K392E (numberings according to Kabat EU index). In a furtherembodiment a first CH3 domain comprises amino acid mutations L351Y,Y407A and a second CH3 domain comprises amino acid mutations T366V.K409F. In a further embodiment, a first CH3 domain comprises amino acidmutation Y407A and a second CH3 domain comprises amino acid mutationsT366A, K409F. In a further embodiment, the second CH3 domain furthercomprises amino acid mutations K392E, T411 E, D399R and S400R(numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2011/143545 is used alternatively. e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409(numbering according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366W and a second CH3 domain comprisesamino acid mutation Y407A. In one embodiment, a first CH3 domaincomprises amino acid mutation T366Y and a second CH3 domain comprisesamino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment, the bispecific antigen binding molecule or its Fcdomain is of IgG₂ subclass and the heterodimerization approach describedin WO 2010/129304 is used alternatively.

In an alternative embodiment, a modification promoting association ofthe first and the second subunit of the Fc domain comprises amodification mediating electrostatic steering effects, e.g. as describedin PCT publication WO 2009/089004. Generally, this method involvesreplacement of one or more amino acid residues at the interface of thetwo Fc domain subunits by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such embodiment, a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),preferably K392D or N392D) and a second CH3 domain comprises amino acidsubstitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K, and more preferably D399K and E356K). In a furtherembodiment, the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negatively charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (allnumberings according to Kabat EU index).

In yet a further embodiment, the heterodimerization approach describedin WO 2007/147901 is used alternatively. In one embodiment, a first CH3domain comprises amino acid mutations K253E, D282K, and K322D and asecond CH3 domain comprises amino acid mutations D239K, E240K, and K292D(numberings according to Kabat EU index).

In still another embodiment, the heterodimerization approach describedin WO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises aminoacid substitutions K392D and K409D, and the second subunit of the Fcdomain comprises amino acid substitutions D356K and D399K (numberingaccording to Kabat EU index).

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the bispecific antigen binding molecule (or theantibody) favorable pharmacokinetic properties, including a long serumhalf-life which contributes to good accumulation in the target tissueand a favorable tissue-blood distribution ratio. At the same time itmay, however, lead to undesirable targeting of the bispecific antigenbinding molecule (or the antibody) to cells expressing Fc receptorsrather than to the preferred antigen-bearing cells. Moreover, theco-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the T cell activating properties(e.g. in embodiments of the bispecific antigen binding molecule whereinthe second antigen binding moiety binds to an activating T cell antigen)and the long half-life of the bispecific antigen binding molecule,results in excessive activation of cytokine receptors and severe sideeffects upon systemic administration. Activation of (Fcreceptor-bearing) immune cells other than T cells may even reduceefficacy of the bispecific antigen binding molecule (particularly abispecific antigen binding molecule wherein the second antigen bindingmoiety binds to an activating T cell antigen) due to the potentialdestruction of T cells e.g. by NK cells.

Accordingly, in particular embodiments, the Fc domain of the bispecificantigen binding molecule according to the invention exhibits reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a native IgG₁ Fc domain. In one such embodiment the Fcdomain (or the bispecific antigen binding molecule comprising said Fcdomain) exhibits less than 50%, preferably less than 20%, morepreferably less than 10% and most preferably less than 5% of the bindingaffinity to an Fc receptor, as compared to a native IgG₁ Fc domain (or abispecific antigen binding molecule comprising a native IgG₁ Fc domain),and/or less than 50%, preferably less than 20%, more preferably lessthan 10% and most preferably less than 5% of the effector function, ascompared to a native IgG₁ Fc domain (or a bispecific antigen bindingmolecule comprising a native IgG₁ Fc domain). In one embodiment, the Fcdomain (or the bispecific antigen binding molecule comprising said Fcdomain) does not substantially bind to an Fc receptor and/or induceeffector function. In a particular embodiment the Fc receptor is an Fcγreceptor. In one embodiment the Fc receptor is a human Fc receptor. Inone embodiment the Fc receptor is an activating Fc receptor. In aspecific embodiment the Fc receptor is an activating human Fcγ receptor,more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specificallyhuman FcγRIIIa. In one embodiment the effector function is one or moreselected from the group of CDC, ADCC, ADCP, and cytokine secretion. In aparticular embodiment, the effector function is ADCC. In one embodiment,the Fc domain exhibits substantially similar binding affinity toneonatal Fc receptor (FcRn), as compared to a native IgG₁ Fc domain.Substantially similar binding to FcRn is achieved when the Fc domain (orthe bispecific antigen binding molecule comprising said Fc domain)exhibits greater than about 70%, particularly greater than about 80%,more particularly greater than about 90% of the binding affinity of anative IgG₁ Fc domain (or the bispecific antigen binding moleculecomprising a native IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the bispecific antigen binding molecule comprises one ormore amino acid mutation that reduces the binding affinity of the Fcdomain to an Fc receptor and/or effector function. Typically, the sameone or more amino acid mutation is present in each of the two subunitsof the Fc domain. In one embodiment, the amino acid mutation reduces thebinding affinity of the Fc domain to an Fc receptor. In one embodiment,the amino acid mutation reduces the binding affinity of the Fc domain toan Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.In embodiments where there is more than one amino acid mutation thatreduces the binding affinity of the Fc domain to the Fc receptor, thecombination of these amino acid mutations may reduce the bindingaffinity of the Fc domain to an Fc receptor by at least 10-fold, atleast 20-fold, or even at least 50-fold. In one embodiment thebispecific antigen binding molecule comprising an engineered Fc domainexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared to abispecific antigen binding molecule comprising a non-engineered Fcdomain. In a particular embodiment, the Fc receptor is an Fcγ receptor.In some embodiments, the Fc receptor is a human Fc receptor. In someembodiments, the Fc receptor is an activating Fc receptor. In a specificembodiment, the Fc receptor is an activating human Fcγ receptor, morespecifically human FcγRIIIa, Fc-γRI or FcγRIIa, most specifically humanFcγRIIIa. Preferably, binding to each of these receptors is reduced. Insome embodiments, binding affinity to a complement component,specifically binding affinity to C1q, is also reduced. In oneembodiment, binding affinity to neonatal Fc receptor (FcRn) is notreduced. Substantially similar binding to FcRn, i.e. preservation of thebinding affinity of the Fc domain to said receptor, is achieved when theFc domain (or the bispecific antigen binding molecule comprising said Fcdomain) exhibits greater than about 70% of the binding affinity of anon-engineered form of the Fc domain (or the bispecific antigen bindingmolecule comprising said non-engineered form of the Fc domain) to FcRn.The Fc domain, or bispecific antigen binding molecules of the inventioncomprising said Fc domain, may exhibit greater than about 80% and evengreater than about 90% of such affinity. In certain embodiments, the Fcdomain of the bispecific antigen binding molecule is engineered to havereduced effector function, as compared to a non-engineered Fc domain.The reduced effector function can include, but is not limited to, one ormore of the following: reduced complement dependent cytotoxicity (CDC),reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming. In one embodiment, the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment, the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a bispecific antigen binding moleculecomprising a non-engineered Fc domain).

In one embodiment, the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment, the Fc domain comprisesan amino acid substitution at a position selected from the group ofE233, L234, L235, N297, P331 and P329 (numberings according to Kabat EUindex). In a more specific embodiment, the Fc domain comprises an aminoacid substitution at a position selected from the group of L234, L235and P329 (numberings according to Kabat EU index). In some embodiments,the Fc domain comprises the amino acid substitutions L234A and L235A(numberings according to Kabat EU index). In one such embodiment, the Fcdomain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain. In oneembodiment, the Fc domain comprises an amino acid substitution atposition P329. In a more specific embodiment, the amino acidsubstitution is P329A or P329G, particularly P329G (numberings accordingto Kabat EU index). In one embodiment, the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionat a position selected from E233, L234, L235, N297 and P331 (numberingsaccording to Kabat EU index). In a more specific embodiment, the furtheramino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D orP331S. In particular embodiments, the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235 (numberings according toKabat EU index). In more particular embodiments, the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA”or “LALAPG”). Specifically, in particular embodiments, each subunit ofthe Fc domain comprises the amino acid substitutions L234A, L235A andP329G (Kabat EU index numbering), i.e. in each of the first and thesecond subunit of the Fc domain the leucine residue at position 234 isreplaced with an alanine residue (L234A), the leucine residue atposition 235 is replaced with an alanine residue (L235A) and the prolineresidue at position 329 is replaced by a glycine residue (P329G)(numbering according to Kabat EU index).

-   -   In one such embodiment, the Fc domain is an IgG₁ Fc domain,        particularly a human IgG₁ Fc domain. The “P329G LALA”        combination of amino acid substitutions almost completely        abolishes Fcγ receptor (as well as complement) binding of a        human IgG₁ Fc domain, as described in PCT publication no. WO        2012/130831, which is incorporated herein by reference in its        entirety. WO 2012/130831 also describes methods of preparing        such mutant Fc domains and methods for determining its        properties such as Fc receptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments, the Fc domain of the bispecific antigen bindingmolecules of the invention is an IgG₄ Fc domain, particularly a humanIgG₄ Fc domain. In one embodiment, the IgG₄ Fc domain comprises aminoacid substitutions at position S228, specifically the amino acidsubstitution S228P (numberings according to Kabat EU index). To furtherreduce its binding affinity to an Fc receptor and/or its effectorfunction, in one embodiment, the IgG₄ Fc domain comprises an amino acidsubstitution at position L235, specifically the amino acid substitutionL235E (numberings according to Kabat EU index). In another embodiment,the IgG₄ Fc domain comprises an amino acid substitution at positionP329, specifically the amino acid substitution P329G (numberingsaccording to Kabat EU index). In a particular embodiment, the IgG₄ Fcdomain comprises amino acid substitutions at positions S228, L235 andP329, specifically amino acid substitutions S228P, L235E and P329G(numberings according to Kabat EU index). Such IgG₄ Fc domain mutantsand their Fcγ receptor binding properties are described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety.

In a particular embodiment, the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG₄ Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G (numberings according to Kabat EU index).

In certain embodiments, N-glycosylation of the Fc domain has beeneliminated. In one such embodiment, the Fc domain comprises an aminoacid mutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D)(numberings according to Kabat EU index).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056) (numberings according to Kabat EU index). SuchFc mutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. Alternatively, binding affinity ofFc domains or bispecific antigen binding molecules comprising an Fcdomain for Fc receptors may be evaluated using cell lines known toexpress particular Fc receptors, such as human NK cells expressingFcγIIIa receptor.

Effector function of an Fc domain, or a bispecific antigen bindingmolecule comprising an Fc domain, can be measured by methods known inthe art. Examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362; Hellstromet al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al.,Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.); and CytoTox 96® non-radioactivecytotoxicity assay (Promega. Madison. Wis.)). Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively. or additionally. ADCC activityof the molecule of interest may be assessed in vivo, e.g. in a animalmodel such as that disclosed in Clynes et al., Proc Natl Acad Sci USA95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the Fc domain, or the bispecificantigen binding molecule comprising the Fc domain, is able to bind C1qand hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., JImmunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052(2003); and Cragg and Glennie. Blood 103, 2738-2743 (2004)).

FcRn binding and in vivo clearance/half life determinations can also beperformed using methods known in the art (see, e.g., Petkova, S. B. etal., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929).

Polynucleotides

The invention further provides isolated polynucleotides encoding anantibody or bispecific antigen binding molecule as described herein or afragment thereof. In some embodiments, said fragment is an antigenbinding fragment.

The polynucleotides encoding antibodies or bispecific antigen bindingmolecules of the invention may be expressed as a single polynucleotidethat encodes the entire antibody or bispecific antigen binding moleculeor as multiple (e.g., two or more) polynucleotides that areco-expressed.

Polypeptides encoded by polynucleotides that are co-expressed mayassociate through, e.g., disulfide bonds or other means to form afunctional antibody or bispecific antigen binding molecule. For example,the light chain portion of an antibody or bispecific antigen bindingmolecule may be encoded by a separate polynucleotide from the portion ofthe antibody or bispecific antigen binding molecule comprising the heavychain of the antibody or bispecific antigen binding molecule. Whenco-expressed, the heavy chain polypeptides will associate with the lightchain polypeptides to form the antibody or bispecific antigen bindingmolecule. In another example, the portion of the antibody or bispecificantigen binding molecule comprising one of the two Fc domain subunitsand optionally (part of) one or more Fab molecules could be encoded by aseparate polynucleotide from the portion of the antibody or bispecificantigen binding molecule comprising the other of the two Fc domainsubunits and optionally (part of) a Fab molecule. When co-expressed, theFc domain subunits will associate to form the Fc domain.

In some embodiments, the isolated polynucleotide encodes the entireantibody or bispecific antigen binding molecule according to theinvention as described herein. In other embodiments, the isolatedpolynucleotide encodes a polypeptide comprised in the antibody orbispecific antigen binding molecule according to the invention asdescribed herein.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

Antibodies or bispecific antigen binding molecules of the invention maybe obtained, for example, by solid-state peptide synthesis (e.g.Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding the antibodyor bispecific antigen binding molecule (fragment), e.g., as describedabove, is isolated and inserted into one or more vectors for furthercloning and/or expression in a host cell. Such polynucleotide may bereadily isolated and sequenced using conventional procedures. In oneembodiment a vector, preferably an expression vector, comprising one ormore of the polynucleotides of the invention is provided. Methods whichare well known to those skilled in the art can be used to constructexpression vectors containing the coding sequence of an antibody orbispecific antigen binding molecule (fragment) along with appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL,Cold Spring Harbor Laboratory. N.Y. (1989); and Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and WileyInterscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe antibody or bispecific antigen binding molecule (fragment) (i.e. thecoding region) is cloned in operable association with a promoter and/orother transcription or translation control elements. As used herein, a“coding region” is a portion of nucleic acid which consists of codonstranslated into amino acids. Although a “stop codon” (TAG, TGA, or TAA)is not translated into an amino acid, it may be considered to be part ofa coding region, if present, but any flanking sequences, for examplepromoters, ribosome binding sites, transcriptional terminators, introns,5′ and 3′ untranslated regions, and the like, are not part of a codingregion. Two or more coding regions can be present in a singlepolynucleotide construct, e.g. on a single vector, or in separatepolynucleotide constructs, e.g. on separate (different) vectors.Furthermore, any vector may contain a single coding region, or maycomprise two or more coding regions, e.g. a vector of the presentinvention may encode one or more polypeptides, which are post- orco-translationally separated into the final proteins via proteolyticcleavage. In addition, a vector, polynucleotide, or nucleic acid of theinvention may encode heterologous coding regions, either fused orunfused to a polynucleotide encoding the antibody or bispecific antigenbinding molecule (fragment) of the invention, or variant or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain. An operable association is when a codingregion for a gene product, e.g. a polypeptide, is associated with one ormore regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein. A varietyof transcription control regions are known to those skilled in the art.These include, without limitation, transcription control regions, whichfunction in vertebrate cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (e.g. the immediate earlypromoter, in conjunction with intron-A), simian virus 40 (e.g. the earlypromoter), and retroviruses (such as, e.g. Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas inducible promoters (e.g. promoters inducible tetracyclins).Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from viral systems (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence). Theexpression cassette may also include other features such as an origin ofreplication, and/or chromosome integration elements such as retrovirallong terminal repeats (LTRs), or adeno-associated viral (AAV) invertedterminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the antibody or bispecific antigen binding molecule is desired, DNAencoding a signal sequence may be placed upstream of the nucleic acidencoding an antibody or bispecific antigen binding molecule of theinvention or a fragment thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling theantibody or bispecific antigen binding molecule may be included withinor at the ends of the antibody or bispecific antigen binding molecule(fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) one ormore vector comprising one or more polynucleotide that encodes (part of)an antibody or bispecific antigen binding molecule of the invention. Asused herein, the term “host cell” refers to any kind of cellular systemwhich can be engineered to generate the antibody or bispecific antigenbinding molecule of the invention or fragments thereof. Host cellssuitable for replicating and for supporting expression of antibodies orbispecific antigen binding molecules are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the antibody or bispecific antigen bindingmolecule for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, polypeptides may be produced in bacteria inparticular when glycosylation is not needed. After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for polypeptide-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CV1), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TR1 cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhfr CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77,4216 (1980)); and myeloma cell lines suchas YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian host celllines suitable for protein production, see, e.g., Yazaki and Wu, Methodsin Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003). Host cells include cultured cells, e.g.,mammalian cultured cells, yeast cells, insect cells, bacterial cells andplant cells, to name only a few, but also cells comprised within atransgenic animal, transgenic plant or cultured plant or animal tissue.In one embodiment, the host cell is a eukaryotic cell, preferably amammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a humanembryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain.

In one embodiment, a method of producing an antibody or bispecificantigen binding molecule according to the invention is provided, whereinthe method comprises culturing a host cell comprising a polynucleotideencoding the antibody or bispecific antigen binding molecule, asprovided herein, under conditions suitable for expression of theantibody or bispecific antigen binding molecule, and optionallyrecovering the antibody or bispecific antigen binding molecule from thehost cell (or host cell culture medium).

The components of the bispecific antigen binding molecule (or theantibody) of the invention may be genetically fused to each other. Thebispecific antigen binding molecule can be designed such that itscomponents are fused directly to each other or indirectly through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Examples of linker sequences between differentcomponents of bispecific antigen binding molecules are provided herein.Additional sequences may also be included to incorporate a cleavage siteto separate the individual components of the fusion if desired, forexample an endopeptidase recognition sequence.

The antibody or bispecific antigen binding molecule of the inventiongenerally comprise at least an antibody variable region capable ofbinding an antigenic determinant. Variable regions can form part of andbe derived from naturally or non-naturally occurring antibodies andfragments thereof. Methods to produce polyclonal antibodies andmonoclonal antibodies are well known in the art (see e.g. Harlow andLane, “Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory,1988). Non-naturally occurring antibodies can be constructed using solidphase-peptide synthesis, can be produced recombinantly (e.g. asdescribed in U.S. Pat. No. 4,186,567) or can be obtained, for example,by screening combinatorial libraries comprising variable heavy chainsand variable light chains (see e.g. U.S. Pat. No. 5,969,108 toMcCafferty).

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region may be used in the antibody or bispecificantigen binding molecule of the invention. Non-limiting antibodies,antibody fragments, antigen binding domains or variable regions usefulin the present invention can be of murine, primate, or human origin. Ifthe antibody or bispecific antigen binding molecule is intended forhuman use, a chimeric form of antibody may be used wherein the constantregions of the antibody are from a human. A humanized or fully humanform of the antibody can also be prepared in accordance with methodswell known in the art (see e. g. U.S. Pat. No. 5,565,332 to Winter).Humanization may be achieved by various methods including, but notlimited to (a) grafting the non-human (e.g., donor antibody) CDRs ontohuman (e.g. recipient antibody) framework and constant regions with orwithout retention of critical framework residues (e.g. those that areimportant for retaining good antigen binding affinity or antibodyfunctions), (b) grafting only the non-human specificity-determiningregions (SDRs or a-CDRs; the residues critical for the antibody-antigeninteraction) onto human framework and constant regions, or (c)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Humanizedantibodies and methods of making them are reviewed, e.g., in Almagro andFransson, Front. Biosci. 13:1619-1633 (2008), and are further described,e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al.,Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos.5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods36:25-34 (2005) (describing specificity determining region (SDR)grafting); Padlan, Mol. Immunol. 28:489498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling). Human framework regions that maybe used for humanization include but are not limited to: frameworkregions selected using the “best-fit” method (see, e.g., Sims et al. J.Immunol. 151:2296 (1993)); framework regions derived from the consensussequence of human antibodies of a particular subgroup of light or heavychain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993));human mature (somatically mutated) framework regions or human germlineframework regions (see, e.g., Almagro and Fransson, Front. Biosci.13:1619-1633 (2008)); and framework regions derived from screening FRlibraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997)and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr OpinImmunol 20, 450-459 (2008). Human antibodies may be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge. Such animalstypically contain all or a portion of the human immunoglobulin loci,which replace the endogenous immunoglobulin loci, or which are presentextrachromosomally or integrated randomly into the animal's chromosomes.In such transgenic mice, the endogenous immunoglobulin loci havegenerally been inactivated. For review of methods for obtaining humanantibodies from transgenic animals, see Lonberg, Nat. Biotech.23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and6,150,584 describing XENOMOUSE™ technology: U.S. Pat. No. 5,770,429describing HUMAB® technology; U.S. Pat. No. 7,041,870 describing K-MMOUSE® technology, and U.S. Patent Application Publication No. US2007/0061900, describing VELOCIMOUSE® technology). Human variableregions from intact antibodies generated by such animals may be furthermodified. e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boemer et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolation from human antibodylibraries, as described herein.

Antibodies useful in the invention may be isolated by screeningcombinatorial libraries for antibodies with the desired activity oractivities. Methods for screening combinatorial libraries are reviewed,e.g., in Lerner et al. in Nature Reviews 16.498-508 (2016). For example,a variety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are reviewed, e.g., inFrenzel et al. in mAbs 8:1177-1194 (2016); Bazan et al. in HumanVaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. inCritical Reviews in Biotechnology 36:276-289 (2016) as well as inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and in Marks and Bradbury inMethods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa,N.J., 2003).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. in Annual Review ofImmunology 12: 433-455 (1994). Phage typically display antibodyfragments, either as single-chain Fv (scFv) fragments or as Fabfragments. Libraries from immunized sources provide high-affinityantibodies to the immunogen without the requirement of constructinghybridomas. Alternatively, the naive repertoire can be cloned (e.g.,from human) to provide a single source of antibodies to a wide range ofnon-self and also self antigens without any immunization as described byGriffiths et al. in EMBO Journal 12: 725-734 (1993). Finally, naïvelibraries can also be made synthetically by cloning unrearranged V-genesegments from stem cells, and using PCR primers containing randomsequence to encode the highly variable CDR3 regions and to accomplishrearrangement in vitro, as described by Hoogenboom and Winter in Journalof Molecular Biology 227: 381-388 (1992). Patent publications describinghuman antibody phage libraries include, for example: U.S. Pat. Nos.5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US PatentPublication Nos. 2005/0079574, 2007/0117126, 2007/0237764 and2007/0292936. Further examples of methods known in the art for screeningcombinatorial libraries for antibodies with a desired activity oractivities include ribosome and mRNA display, as well as methods forantibody display and selection on bacteria, mammalian cells, insectcells or yeast cells. Methods for yeast surface display are reviewed,e.g., in Scholler et al. in Methods in Molecular Biology 503:135-56(2012) and in Cherf et al. in Methods in Molecular biology 1319:155-175(2015) as well as in the Zhao et al. in Methods in Molecular Biology889:73-84 (2012). Methods for ribosome display are described, e.g., inHe et al. in Nucleic Acids Research 25:5132-5134 (1997) and in Hanes etal. in PNAS 94:4937-4942 (1997).

Antibodies or bispecific antigen binding molecules prepared as describedherein may be purified by art-known techniques such as high performanceliquid chromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography, size exclusion chromatography, and the like.The actual conditions used to purify a particular protein will depend,in part, on factors such as net charge, hydrophobicity, hydrophilicityetc., and will be apparent to those having skill in the art. Foraffinity chromatography purification, an antibody, ligand, receptor orantigen can be used to which the antibody or bispecific antigen bindingmolecule binds. For example, for affinity chromatography purification ofantibodies or bispecific antigen binding molecules of the invention, amatrix with protein A or protein G may be used. Sequential Protein A orG affinity chromatography and size exclusion chromatography can be usedto isolate an antibody or bispecific antigen binding moleculeessentially as described in the Examples. The purity of the antibody orbispecific antigen binding molecule can be determined by any of avariety of well known analytical methods including gel electrophoresis,high pressure liquid chromatography, and the like.

Assays

Antibodies or bispecific antigen binding molecules provided herein maybe identified, screened for, or characterized for theirphysical/chemical properties and/or biological activities by variousassays known in the art.

Affinity Assays

The affinity of the antibody or bispecific antigen binding molecule foran Fc receptor or a target antigen can be determined for example bysurface plasmon resonance (SPR), using standard instrumentation such asa BIAcore instrument (GE Healthcare), and receptors or target proteinssuch as may be obtained by recombinant expression. Alternatively,binding of antibodies or bispecific antigen binding molecules fordifferent receptors or target antigens may be evaluated using cell linesexpressing the particular receptor or target antigen, for example byflow cytometry (FACS). A specific illustrative and exemplary embodimentfor measuring binding affinity is described in the following.

According to one embodiment. K_(D) is measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C.

To analyze the interaction between the Fc-portion and Fc receptors,His-tagged recombinant Fc-receptor is captured by an anti-Penta Hisantibody (Qiagen) immobilized on CM5 chips and the bispecific constructsare used as analytes. Briefly, carboxymethylated dextran biosensor chips(CM5, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti Penta-His antibody is diluted with 10 mM sodium acetate, pH 5.0, to40 μg/ml before injection at a flow rate of 5 μl/min to achieveapproximately 6500 response units (RU) of coupled protein. Following theinjection of the ligand, 1 M ethanolamine is injected to block unreactedgroups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM.For kinetic measurements, four-fold serial dilutions of the antibody orbispecific antigen binding molecule (range between 500 nM and 4000 nM)are injected in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mMEDTA, 0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate of 30μl/min for 120 s.

To determine the affinity to the target antigen, antibodies orbispecific antigen binding molecules are captured by an anti human Fabspecific antibody (GE Healthcare) that is immobilized on an activatedCM5-sensor chip surface as described for the anti Penta-His antibody.The final amount of coupled protein is approximately 12000 RU. The,antibodies or bispecific antigen binding molecules are captured for 90 sat 300 nM. The target antigens are passed through the flow cells for 180s at a concentration range from 250 to 1000 nM with a flowrate of 30μl/min. The dissociation is monitored for 180 s.

Bulk refractive index differences are corrected for by subtracting theresponse obtained on reference flow cell. The steady state response wasused to derive the dissociation constant K_(D) by non-linear curvefitting of the Langmuir binding isotherm. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

Activity Assays

Biological activity of the bispecific antigen binding molecules (orantibodies) of the invention can be measured by various assays asdescribed in the Examples. Biological activities may for example includethe induction of proliferation of T cells, the induction of signaling inT cells, the induction of expression of activation markers in T cells,the induction of cytokine secretion by T cells, the induction of lysisof target cells such as tumor cells, and the induction of tumorregression and/or the improvement of survival.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the antibodies or bispecific antigen binding moleculesprovided herein, e.g., for use in any of the below therapeutic methods.In one embodiment, a pharmaceutical composition comprises any of theantibodies or bispecific antigen binding molecules provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises any of the antibodies or bispecificantigen binding molecules provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Further provided is a method of producing an antibody or bispecificantigen binding molecule of the invention in a form suitable foradministration in vivo, the method comprising (a) obtaining an antibodyor bispecific antigen binding molecule according to the invention, and(b) formulating the antibody or bispecific antigen binding molecule withat least one pharmaceutically acceptable carrier, whereby a preparationof antibody or bispecific antigen binding molecule is formulated foradministration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of antibody or bispecific antigenbinding molecule dissolved or dispersed in a pharmaceutically acceptablecarrier. The phrases “pharmaceutical or pharmacologically acceptable”refers to molecular entities and compositions that are generallynon-toxic to recipients at the dosages and concentrations employed, i.e.do not produce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of a pharmaceutical composition thatcontains an antibody or bispecific antigen binding molecule andoptionally an additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards or correspondingauthorities in other countries. Preferred compositions are lyophilizedformulations or aqueous solutions. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, buffers, dispersionmedia, coatings, surfactants, antioxidants, preservatives (e.g.antibacterial agents, antifungal agents), isotonic agents, absorptiondelaying agents, salts, preservatives, antioxidants, proteins, drugs,drug stabilizers, polymers, gels, binders, excipients, disintegrationagents, lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

An antibody or bispecific antigen binding molecule of the invention (andany additional therapeutic agent) can be administered by any suitablemeans, including parenteral, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the antibodies or bispecific antigen binding molecules ofthe invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. The solution may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the antibodies or bispecific antigen bindingmolecules may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the antibodies orbispecific antigen binding molecules of the invention in the requiredamount in the appropriate solvent with various of the other ingredientsenumerated below, as required. Sterility may be readily accomplished,e.g., by filtration through sterile filtration membranes. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and/or the other ingredients. In the case of sterile powders forthe preparation of sterile injectable solutions, suspensions oremulsion, the preferred methods of preparation are vacuum-drying orfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered liquid medium thereof. The liquid medium should besuitably buffered if necessary and the liquid diluent first renderedisotonic prior to injection with sufficient saline or glucose. Thecomposition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids: antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride:benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben: catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone: amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA: sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium, metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the antibodies orbispecific antigen binding molecules may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the antibodies or bispecificantigen binding molecules may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Pharmaceutical compositions comprising the antibodies or bispecificantigen binding molecules of the invention may be manufactured by meansof conventional mixing, dissolving, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The antibodies or bispecific antigen binding molecules may be formulatedinto a composition in a free acid or base, neutral or salt form.Pharmaceutically acceptable salts are salts that substantially retainthe biological activity of the free acid or base. These include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or w % bich are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Pharmaceutical salts tend to be more soluble inaqueous and other protic solvents than are the corresponding free baseforms.

Therapeutic Methods and Compositions

Any of the antibodies or bispecific antigen binding molecules providedherein may be used in therapeutic methods. Antibodies or bispecificantigen binding molecules of the invention may be used asimmunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, antibodies or bispecific antigen bindingmolecules of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, antibodies or bispecific antigen binding molecules of theinvention for use as a medicament are provided. In further aspects,antibodies or bispecific antigen binding molecules of the invention foruse in treating a disease are provided. In certain embodiments,antibodies or bispecific antigen binding molecules of the invention foruse in a method of treatment are provided. In one embodiment, theinvention provides an antibody or bispecific antigen binding molecule asdescribed herein for use in the treatment of a disease in an individualin need thereof. In certain embodiments, the invention provides anantibody or bispecific antigen binding molecule for use in a method oftreating an individual having a disease comprising administering to theindividual a therapeutically effective amount of the antibody orbispecific antigen binding molecule. In certain embodiments the diseaseto be treated is a proliferative disorder. Ina particular embodiment thedisease is cancer. In certain embodiments the method further comprisesadministering to the individual a therapeutically effective amount of atleast one additional therapeutic agent, e.g., an anti-cancer agent ifthe disease to be treated is cancer. In further embodiments, theinvention provides an antibody or bispecific antigen binding molecule asdescribed herein for use in inducing lysis of a target cell,particularly a tumor cell. In certain embodiments, the inventionprovides an antibody or bispecific antigen binding molecule for use in amethod of inducing lysis of a target cell, particularly a tumor cell, inan individual comprising administering to the individual an effectiveamount of the antibody or bispecific antigen binding molecule to inducelysis of a target cell. An “individual” according to any of the aboveembodiments is a mammal, preferably a human.

In a further aspect, the invention provides for the use of an antibodyor bispecific antigen binding molecule of the invention in themanufacture or preparation of a medicament. In one embodiment themedicament is for the treatment of a disease in an individual in needthereof. In a further embodiment, the medicament is for use in a methodof treating a disease comprising administering to an individual havingthe disease a therapeutically effective amount of the medicament. Incertain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In oneembodiment, the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In a further embodiment, the medicament is forinducing lysis of a target cell, particularly a tumor cell. In still afurther embodiment, the medicament is for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of themedicament to induce lysis of a target cell. An “individual” accordingto any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such disease a therapeutically effective amount of anantibody or bispecific antigen binding molecule of the invention. In oneembodiment a composition is administered to said individual, comprisingthe antibody or bispecific antigen binding molecule of the invention ina pharmaceutically acceptable form. In certain embodiments the diseaseto be treated is a proliferative disorder. In a particular embodimentthe disease is cancer. In certain embodiments the method furthercomprises administering to the individual a therapeutically effectiveamount of at least one additional therapeutic agent, e.g., ananti-cancer agent if the disease to be treated is cancer. An“individual” according to any of the above embodiments may be a mammal,preferably a human.

In a further aspect, the invention provides a method for inducing lysisof a target cell, particularly a tumor cell. In one embodiment themethod comprises contacting a target cell with an antibody or bispecificantigen binding molecule of the invention in the presence of a T cell,particularly a cytotoxic T cell. In a further aspect, a method forinducing lysis of a target cell, particularly a tumor cell, in anindividual is provided. In one such embodiment, the method comprisesadministering to the individual an effective amount of an antibody orbispecific antigen binding molecule to induce lysis of a target cell. Inone embodiment, an “individual” is a human.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that may be treated using an antibodyor bispecific antigen binding molecule of the present invention include,but are not limited to neoplasms located in the: abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Also included are pre-cancerous conditions or lesionsand cancer metastases. In certain embodiments the cancer is chosen fromthe group consisting of kidney cancer, bladder cancer, skin cancer, lungcancer, colorectal cancer, breast cancer, brain cancer, head and neckcancer and prostate cancer. In one embodiment, the cancer is prostatecancer. A skilled artisan readily recognizes that in many cases theantibody or bispecific antigen binding molecule may not provide a curebut may only provide partial benefit. In some embodiments, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some embodiments, an amount ofantibody or bispecific antigen binding molecule that provides aphysiological change is considered an “effective amount” or a“therapeutically effective amount”. The subject, patient, or individualin need of treatment is typically a mammal, more specifically a human.

In some embodiments, an effective amount of an antibody or bispecificantigen binding molecule of the invention is administered to a cell. Inother embodiments, a therapeutically effective amount of an antibody orbispecific antigen binding molecule of the invention is administered toan individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of anantibody or bispecific antigen binding molecule of the invention (whenused alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the route of administration, the body weight of the patient, the type ofantibody or bispecific antigen binding molecule, the severity and courseof the disease, whether the antibody or bispecific antigen bindingmolecule is administered for preventive or therapeutic purposes,previous or concurrent therapeutic interventions, the patient's clinicalhistory and response to the antibody or bispecific antigen bindingmolecule, and the discretion of the attending physician. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosing schedulesincluding but not limited to single or multiple administrations overvarious time-points, bolus administration, and pulse infusion arecontemplated herein.

The antibody or bispecific antigen binding molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or bispecific antigenbinding molecule can be an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the antibody or bispecific antigen binding moleculewould be in the range from about 0.005 mg/kg to about 10 mg/kg. In othernon-limiting examples, a dose may also comprise from about 1microgram/kg body weight, about 5 microgram/kg body weight, about 10microgram/kg body weight, about 50 microgram/kg body weight, about 100microgram/kg body weight, about 200 microgram/kg body weight, about 350microgram/kg body weight, about 500 microgram/kg body weight, about 1milligram/kg body weight, about 5 milligram/kg body weight, about 10milligram/kg body weight, about 50 milligram/kg body weight, about 100milligram/kg body weight, about 200 milligram/kg body weight, about 350milligram/kg body weight, about 500 milligram/kg body weight, to about1000 mg/kg body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg body weight to about100 mg/kg body weight, about 5 microgram/kg body weight to about 500milligram/kg body weight, etc., can be administered, based on thenumbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently. e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the antibody or bispecific antigen binding molecule). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The antibodies or bispecific antigen binding molecules of the inventionwill generally be used in an amount effective to achieve the intendedpurpose. For use to treat or prevent a disease condition, the antibodiesor bispecific antigen binding molecules of the invention, orpharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data. e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the antibodies or bispecific antigen binding moleculeswhich are sufficient to maintain therapeutic effect. Usual patientdosages for administration by injection range from about 0.1 to 50mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the antibodies or bispecific antigen bindingmolecules may not be related to plasma concentration. One having skillin the art will be able to optimize therapeutically effective localdosages without undue experimentation.

A therapeutically effective dose of the antibodies or bispecific antigenbinding molecules described herein will generally provide therapeuticbenefit without causing substantial toxicity. Toxicity and therapeuticefficacy of an antibody or bispecific antigen binding molecule can bedetermined by standard pharmaceutical procedures in cell culture orexperimental animals. Cell culture assays and animal studies can be usedto determine the LD₅₀ (the dose lethal to 50% of a population) and theED₅₀ (the dose therapeutically effective in 50% of a population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, which can be expressed as the ratio LD₅₀/ED₅₀. Antibodies orbispecific antigen binding molecules that exhibit large therapeuticindices are preferred. In one embodiment, the antibody or bispecificantigen binding molecule according to the present invention exhibits ahigh therapeutic index. The data obtained from cell culture assays andanimal studies can be used in formulating a range of dosages suitablefor use in humans. The dosage lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon a varietyof factors, e.g., the dosage form employed, the route of administrationutilized, the condition of the subject, and the like. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in; The Pharmacological Basis of Therapeutics. Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with antibodies orbispecific antigen binding molecules of the invention would know how andwhen to terminate, interrupt, or adjust administration due to toxicity,organ dysfunction, and the like. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose in the management of the disorder of interest willvary with the severity of the condition to be treated, with the route ofadministration, and the like. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency will also varyaccording to the age, body weight, and response of the individualpatient.

Other Agents and Treatments

The antibodies and bispecific antigen binding molecules of the inventionmay be administered in combination with one or more other agents intherapy. For instance, an antibody or bispecific antigen bindingmolecule of the invention may be co-administered with at least oneadditional therapeutic agent. The term “therapeutic agent” encompassesany agent administered to treat a symptom or disease in an individual inneed of such treatment. Such additional therapeutic agent may compriseany active ingredients suitable for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. In certain embodiments, an additionaltherapeutic agent is an immunomodulatory agent, a cytostatic agent, aninhibitor of cell adhesion, a cytotoxic agent, an activator of cellapoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent. Such other agents are suitably present incombination in amounts that are effective for the purpose intended. Theeffective amount of such other agents depends on the amount of antibodyor bispecific antigen binding molecule used, the type of disorder ortreatment, and other factors discussed above. The antibodies orbispecific antigen binding molecules are generally used in the samedosages and with administration routes as described herein, or aboutfrom 1 to 99% of the dosages described herein, or in any dosage and byany route that is empirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the antibody or bispecific antigen binding molecule ofthe invention can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/or adjuvant.Antibodies or bispecific antigen binding molecules of the invention mayalso be used in combination with radiation therapy.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or bispecific antigen binding molecule of theinvention. The label or package insert indicates that the composition isused for treating the condition of choice. Moreover, the article ofmanufacture may comprise (a) a first container with a compositioncontained therein, wherein the composition comprises an antibody orbispecific antigen binding molecule of the invention; and (b) a secondcontainer with a composition contained therein, wherein the compositioncomprises a further cytotoxic or otherwise therapeutic agent. Thearticle of manufacture in this embodiment of the invention may furthercomprise a package insert indicating that the compositions can be usedto treat a particular condition. Alternatively, or additionally, thearticle of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringers solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-STEAP-1 antibodies providedherein is useful for detecting the presence of STEAP-1 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as prostate tissue.

In one embodiment, an anti-STEAP-1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of STEAP-1 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-STEAP-1 antibody as described herein underconditions permissive for binding of the anti-STEAP-1 antibody toSTEAP-1, and detecting whether a complex is formed between theanti-STEAP-1 antibody and STEAP-1. Such method may be an in vitro or invivo method. In one embodiment, an anti-STEAP-1 antibody is used toselect subjects eligible for therapy with an anti-STEAP-1 antibody, e.g.where STEAP-1 is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer, particularly prostate cancer.

In certain embodiments, labeled anti-STEAP-1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases. e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthala-rinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

Amino Acid Sequences Amino Acid Sequence SEQ ID NO STEAP-1 SDYAWN  1HCDR1 STEAP-2 YSINSGSTSYNPSLKS  2 HCDR2 STEAP-1 ERYNYDY DD YYYAMDY  3HCDR3 (DD) STEAP-1 ERNYDY ED YYYAMDY  4 HCDR3 (ED) STEAP-1 ERNYDY DEYYYAMDY  5 HCDR3 (DE) STEAP-1 ERNYDY EE YYYAMDY  6 HCDR3 (EE) STEAP-1KSSQSLLYRSNQKNYLA  7 LCDR1 STEAP-1 WASTRES  8 LCDR2 STEAP-1 QQYYNYPRT  9LCDR3 STEAP-1 EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 10VH (DD) VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC ARERNYDY DDYYYAMDYWGQGTLVTVSS STEAP-1EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 11 VH (ED)VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC ARERNYDY EDYYYAMDYWGQGTLVTVSS STEAP-1EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 12 VH (DE)VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC ARERNYDY DEYYYAMDYWGQGTLVTVSS STEAP-1EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 13 VH (EE)VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC ARERNYDY EEYYYAMDYWGQGTLVTVSS STEAP-1DIQMTQSPSSLSASVGDRVTITCKSSQSLLYRSNQKNYLAWYQQKPGK 14 VLAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ YYNYPRTFGQGTKVEIK CD3TYAMN 15 HCDR1 CD3 RIRSKYNNYATYYADSVKG 16 HCDR2 CD3 HGNFGNSYVSWFAY 17HCDR3 CD3 GSSTGAVTTSNYAN 18 LCDR1 CD3 GTNKRAP 19 LCDR2 CD3 ALWYSNLWV 20LCDR3 CD3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWV 21SRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS CD3 VLQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRG 22LIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSN LWVFGGGTKLTVL hSTEAP-1MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQ 23TAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEIC SQL hCD3MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILT 24CPQYGPSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIR KGQRDLYSGLNQRRI cynoCD3MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILT 25CSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSG LNQRRI Molecule AEVLQVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 26 (STEAP-1VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC VH-(CH1(EE)- ARERNYDYDD YYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG Fc(knob, PGLALA))GTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSLCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPMolecule A EVLQVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 27 (STEAP-1VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC VH-CH1(EE)-CD3 ARERNYDYDD YYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG VL-CH1-Fc(knob),GTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSLGYSLSSV PGLALA))VTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSP Molecule BEVQLVESGGGLVQPGGSLRLSCASSGYSITSDYQWNWVRQAPGKGLEW 28 (STEAP-1VGYSINSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC VH-CH1(EE)- ARERNYDY EDYYYAMDYWGQGLTVTVSSASTKGPSVFPLAPSSKSTSG Fc(knob),GTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV PGLALA))VTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCACKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPMolecule B EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYSWNWVRQAPGKGLEW 29 (STEAP-1VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC VH-CH1(EE)-CD3 ARERNYDYED YYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG VL-CH1-Fc(knob),GTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV PGLALA))VTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKA LSLSP Molecule CEVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 30 (STEAP-1VGYISNSGSTYTYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYY VH-CH1(EE)- CARERNYDYDE YYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS Fc(knob),GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS PGLALA))VVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPMolecule C EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 31 (STEAP-1VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC VH-CH1(EE)-CD3 ARERNYDYDE YYYANDYWGQGTLVTVSSASTKGPHTFPAVLQSSGLYS VL-CH1-Fc(knob),LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGG PGLALA))GSWAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVDLSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSP Molecule DEVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 32 (STEAP-1VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC VH(CH1(EE)- ARERNYDY EEYYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG Fc(knob),GTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV PGLALA))VTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPMolecule D EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEW 33 (STEAP-1VGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYC VH-VH1(EE)-CD3 ARERNYDYEE YYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG VL-CH1-Fc(knob),GTAALGCLVEDYFPEPVTVSWNSGLATSGVHTFPAVLQSSGLYSLSSV PGLALA))VTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRPTEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSP Molecule A-DDIQMTQSPSSLSASVGDRVTITCKSSQSLLYRSNQKNYLAWYQQKPGK 34 (STEAP-1APKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ VL-CL(RK))YYNYPRTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Molecule A-DEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWV 35 (CD3 VH-CL)SRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLMQNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hIgG1 FcDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH 36 regionEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP linker GGGGSGGGGS 37 linkerDGGGGSGGGGS 38 Human kappa CLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ 39 domainSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGECHuman lambda CL QPKAAPSVTLFPPSSEEQANKATLVCLISDFYPGAVTVAWKADSSPVK 40domain AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTECSHuman IgG1 heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS 41chain constant GVHTFPAVLQSSLGYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK regionKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC (CH1—CH2—CH3)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Generation of Constructs and Tools

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al, Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory press, Cold spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

General information regarding the nucleotide sequences of humanimmunoglobulin light and heavy chains is given in; Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991).

Gene Synthesis

Desired gene segments, where required, were either generated by PCRusing appropriate templates or were synthesized at Geneart A G(Regensburg, Germany) from synthetic oligonucleotides and PCR productsby automated gene synthesis. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardcloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow subcloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells.

DNA Sequencing

DNA sequences were determined by double strand sequencing

Cloning of Anti-STEAP1/Anti-CD3 T Cell Bispecific (TCB) Antibodies

The variable domains of vandortuzumab were used for the generation ofvarious STEAP1-specific T cell bispecific (TCB) antibody variants. Forthe generation of the respective expression plasmids, the variableregion sequences of vandortuzumab (SEQ ID NOs 10 and 14) or variantsthereof were used and sub-cloned in frame with the respective constantregions which are pre-inserted in the respective recipient mammalianexpression vector. A schematic illustration of the resulting moleculesis shown in FIG. 2.

Preparation of Anti-STEAP1/Anti-CD3 T Cell Bispecific (TCB) Antibodies

The following molecules were prepared, a schematic illustration thereofis provided in FIG. 2:

-   -   A. Molecule A: 2+1 IgG CrossFab “inverted” (CD3 binder        C-terminal to STEAP1 binder), with charge modifications (VH/VL        exchange in CD3 binder, charge modification in STEAP1 binder)        (SEQ ID NOs 26, 27, 34 and 35)    -   B. Molecule B: 2+1 IgG CrossFab “inverted” (CD3 binder        C-terminal to STEAP1 binder), with charge modifications (VH/VL        exchange in CD3 binder, charge modification in STEAP1 binder;        D100aE mutation in both STEAP1 binding moieties) (SEQ ID NOs 28,        29, 34 and 35)    -   C. Molecule C: 2+1 IgG CrossFab “inverted” (CD3 binder        C-terminal to STEAP1 binder), with charge modifications (VH/VL        exchange in CD3 binder, charge modification in STEAP1 binder;        D100bE mutation in both STEAP1 binding moieties) (SEQ ID NOs 30,        31, 34 and 35)    -   D. Molecule D: 2+1 IgG CrossFab “inverted” (CD3 binder        C-terminal to STEAP1 binder), with charge modifications (VH/VL        exchange in CD3 binder, charge modification in STEAP1 binder;        D100aE/D100bE mutations in both STEAP1 binding moieties) (SEQ ID        NOs 32, 33, 34 and 35)

Expression of the above-mentioned molecules was either driven by achimeric MPSV promoter or a CMV promoter. Polyadenylation was driven bya synthetic polyA signal sequence located at the 3′ end of the CDS. Inaddition, each vector contained an EBV OriP sequence for autosomalreplication.

For the production of all constructs, HEK293-EBNA cells growing insuspension were co-transfected with the respective expression vectorsusing polyethylenimine as a transfection reagent. As such, for theproduction of all “2+1 IgG CrossFab” constructs, the correspondingexpression vectors were co-transfected in a 1:2:1:1 ratio (“vector heavychain (VH—CH1-VL-CH1-CH2-CH3)”: “vector light chain (VL-CL)”: “vectorheavy chain (VH—CH1-CH2-CH3)”: “vector light chain (VH-CL)”).

HEK293 EBNA cells were cultivated in suspension in serum free Excellculture medium containing 6 mM L-Glutamine and 250 mg/I G418. For theproduction in 600 ml tubespin flasks (max, working volume 400 mL) 600million HEK293 EBNA cells were seeded 24 hours before transfection.Before transfection, cells were centrifuged for 5 min by 210×g andsupernatant was replaced by pre-warmed 20 ml CD CHO medium. Expressionvectors were mixed in 20 ml CD CHO medium to a final amount of 400 μgDNA. After addition of 1080 μl PEI solution (2.7 μg/ml), the medium wasvortexed for 15 s and subsequently incubated for 10 min at roomtemperature. Afterwards, cells were mixed with the DNA/PEI solution,transferred to a 600 ml tubespin flask and incubated for 3 hours at 37°C. in an incubator with a humidified 5% CO₂ atmosphere. After thisincubation step, 360 ml Excell medium containing 6 mM L-Glutamine, 5 g/LPepsoy, and 1.0 mM VPA was added and cells were cultivated for 24 hours.One day after transfection, 7% Feed 7 is added. After 7 days ofcultivation, supernatant was collected for purification bycentrifugation for 20-30 min at 3600×g (Sigma 8K centrifuge), thesolution was sterile filtered (0.22 μm filter) and sodium azide wasadded to a final concentration of 0.01% w/v, and kept at 4° C.

All molecules were purified from cell culture supernatants by Protein Aaffinity chromatography, followed by a size exclusion chromatographicstep. For affinity chromatography, supernatant was loaded on a HiTrapProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 25 ml 20mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Unbound protein wasremoved by washing with at least 10 column volumes 20 mM sodiumphosphate, 20 mM sodium citrate, pH 7.5. Target protein was eluted in 6column volumes 20 mM sodium citrate, 100 mM sodium chloride, 100 mMglycine, pH 3.0. Protein solution was neutralized by adding 1/10 volumeof 0.5 M sodium phosphate, pH 8.0. For in-process analytics afterProtein A chromatography, the purity and molecular weight of themolecules in the single fractions were analyzed by SDS-PAGE in theabsence of a reducing agent and stained with Coomassie (InstantBlue™from Expedeon). The NuPAGE® Pre-Cast gel system (4-12% Bis-Tris,Invitrogen, USA) was used according to the manufacturer's instruction.Selected fractions of the target protein were concentrated and filtratedprior to loading on a HiLoad Superdex 200 column (GE Healthcare)equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0, 0.01%Tween20.

The protein concentration of the purified protein samples was determinedby the optical density (OD) at 280 nm using the molar extinctioncoefficient which was calculated on the basis of the amino acidsequence. In addition, mass spectrometry analysis of all molecules wasperformed in order to confirm their identity.

Generation of a STEAP1 Expressing CHO-K1 Cell Line

A gene encoding full-length human STEAP1 was subcloned into mammalianexpression vector. The plasmid was transfected into CHO-K1 (ATCCCRL-9618) cells using Lipofectamine LTX Reagent according to themanufacturer's protocol (Invitrogen, #15338100). Stably transfectedSTEAP1-positive CHO cells were maintained in DMEM/F-12 medium (Gibco,#11320033) supplemented with 10% fetal bovine serum (Gibco, #16140063)and 1% GlutaMAX Supplement (Gibco; #31331-028). Two days aftertransfection, puromycin (Invivogen; #ant-pr-1) was added to 6 μg/mL andthe cells were cultured for several passages. After initial selection,the cells with the highest cell surface expression of STEAP1 were sortedby BD FACSAria II cell sorter (BD Biosciences) and cultured to establishstable cell clones. The expression level and stability was confirmed byFACS analysis using vandortuzumab and PerCP-conjugated Fc gamma-specificgoat anti-human IgG (Jackson ImmunoResearch, #109-126-097) as secondaryantibody over a period of 4 weeks.

Example 2 Generation and Characterization of Vandortuzumab-BasedSequence Variants

Sequence Analysis of Vandortuzumab

Modifications like asparagine deamidation, aspartate isomerization,succinimide formation, and tryptophane oxidation are typicaldegradations for recombinant antibodies and can affect both in vitrostability and in vivo biological functions. A computational analysis ofthe vandortuzumab CDR sequences (SEQ ID NOs 1, 2, 3, 7, 8 and 9,according to Kabat) was performed in order to screen for the presence ofpotential amino acid sequence patterns that are prone to aspartateisomerization, asparagine deamidation or succinimide formation.Furthermore, CDR regions were analyzed for the presence of tryptophanesthat have the potential to oxidize. As shown in FIG. 3A and FIG. 3B, theanalysis of the vandortuzumab sequences revealed potential hotspots inthe CDR regions of the variable domains of both heavy and light chains.

Generation of Variants of the Vandortuzumab Sequence (Molecules B-D)

In order to prepare an anti-STEAP1 antibody with minimal iso-aspartateand succinimide formation and optimal stability, several variants of thevandortuzumab sequence were generated with a modified HCDR3 sequence. Inparticular, the aspartate residues at position 100a and 100b (Kabatnumbering) were replaced by glutamate either individually or incombination (SEQ ID NOs 4, 5 and 6) and the resulting plasmids (SEQ IDNOs 28, 29, 34 and 35 (mutation D100aE, Molecule B); SEQ ID NOs 30, 31,34 and 35 (mutation D100bE, Molecule C); SEQ ID NOs 32-35 (mutationsD100aE/D100bE, Molecule D)) were generated for the expression of therespective TCB antibody molecules.

Chemical Degradation Test

In order to confirm that the introduced mutations eliminate thepredicted hotspot in HCDR3, increase the stability and prevent loss ofbinding potency of the anti-STEAP1 antibodies, all constructs (MoleculesA-D) were split into two aliquots, re-buffered into 20 mM His/HisCl, 140mM NaCl, pH 6.0 or into PBS, pH 7.4, respectively, and incubated at 40°C. (His/NaCl) or 37° C. (PBS). In addition, a control sample was storedat −80° C. Incubation at pH 7.4 reflects the exposure of the molecule tothe situation in the blood plasma and allows drawing conclusions aboutthe stability of the molecule in vivo. In contrast, buffer formulationswith reduced pH (here pH 6) are more suitable for long term storage ofantibody-based constructs and stress tests under these conditions arepredictive for the shelf life of a molecule.

After an incubation period of 28 days, samples were analyzed andcompared for the binding potency (relative active concentration) of theSTEAP1-binding moieties. This analysis was performed indirectly by massspectrometry and a cell-based ELISA.

Characterization of Stressed Molecule A-D by Mass Spectrometry

In order to identify stress-induced protein degradation at predictedpositions within the CDRs of vandortuzumab and variants thereof, massspectrometry of Molecules A-D was performed, 80 μg reference andstressed protein samples were denatured and reduced for 1 h in 124.5 μl100 mM Tris, 5.6 M guanidinium hydrochloride, 10 mM TCEP(tris(2-carboxyethyl)phosphine (Pierce Protein Biology Products), pH 6.0at 37° C. Buffer was exchanged to 20 mM histidine chloride, 0.5 mM TCEP,pH 6.0 in 0.5 mL Zeba Spin Desalting Columns (Pierce Protein BiologyProducts). Protein samples were digested overnight at 37° C. afteraddition of 0.05 μg trypsin (Promega) per μg protein in a final volumeof 140 μL. Digestion was stopped by addition of 7 μL of 10% formic acid(FA) solution.

The digested samples were stored at −80° C. until use. Analysis wasperformed by UHPLC-MS/MS using a nanoAcquity UPLC (Waters) and anOrbitrap Fusion mass spectrometer (Thermo Fisher Scientific). About 2.4μg digested fusion protein was injected in 5 μL. Chromatographicseparation was performed by reversed-phase on a Acquity BEH300 C18column, 1×150 mm, 1.7 μm, 300 Å (Waters) using a flow rate of 60 μL/min.The mobile phase A and B contained 0.1% (v/v) formic acid in UPLC gradewater and acetonitrile, respectively. A column temperature of 50° C. wasused and a gradient of 1% to 40% mobile phase B over 90 min was applied.The Orbitrap Fusion was used in the data-dependent mode. Essential MSsettings were: ionization (spray voltage: 3.6 kV, ion transfer tube:250° C., vaporizer: 100° C.), full MS (AGC: 2×105, resolution: 12×104,m/z range: 300-2000, maximum injection time: 100 ms); MS/MS (AGC: 1×104,maximum injection time: 100 ms, isolation width: 2 Da). Normalizedcollision energy was set to 35%.

Peptide mapping was applied to quantify deamidation, isomerization andoxidation levels of the predicted hotspots (N53 (HCDR2), N97 (HCDR3),D100a (HCDR3) and W50 (LCDR2) (Kabat numbering)) for Molecules A-D.

In the peptides harboring the HCDR3 region, no deamidation orsuccinimide formation at position N97 was detected in either of theMolecules A-D, at either condition (data not shown). However, stressexposure at pH 6 resulted in different levels of aspartate degradationin the same HCDR3 peptide that also harbors the predicted hotspotaspartate 100a. Different levels of succinimide and iso-aspartateformation were detected after 4 weeks in His/NaCl pH 6.0 at 40° C. inthe respective tryptic peptides of the four tested molecules (Table 1).After stress exposure at pH 6.0, the highest total levels were detectedin Molecule A. Introduction of mutation of D100a→E100a in Molecule B aswell as D100b→E100b in Molecule C lead to significant decreases of thesuccinimide level. However, combination of both mutations(D100aE/D100bE) (Molecule D) strongly reduced the succinimide levels to3%, a negligible amount for this long period of stress exposure.Furthermore, no iso-aspartate was detected in Molecule D. The resultsindicate that both aspartates (D100a and D100b) in Molecule A and eitherone of the aspartates (D100a or D100b) in Molecules C or B contribute tothe total succinimide and iso-aspartate levels found after stress. Inaddition, no protein degradation at all was detected in HCDR3 ofMolecule D after stress exposure at pH 7.4 confirming the integrity ofthis newly designed sequence variant.

TABLE 1 Relative quantification of protein degradation in HCDR3.4 weeks in His/NaCl 4 weeks in PBS His/NaCl Tryptic peptidepH 6.0 at 40° C. pH 7.4 at 37° C. pH 6.0 control with indicated iso-aspsuccinimide iso-asp succinimide iso-asp succinimide Sample mutations*[%] [%] [%] [%] [%] [%] Molecule A ERNYDYDDYYYAMDY 1.4 12.7 not 5.3 not4.2 WGQGTLVTVSSASTK detec-ted detec-ted Molecule B ERNYDY E DYYYAMDY 5.35.0 2.7 0.9 2.8 1.1 WGQGTLVTVSSASTK Molecule C ERNYDYD E YYYAMDY 1.2 6.20.8 1.3 0.04 1.5 WGQGTLVTVSSASTK Molecule D ERNYDY EE YYYAMDY not 3.0not not not not WGQGTLVTVSSASTK detec-ted detec-ted detec-ted detec-teddetec-ted *Mutated positions bold and underlined

Analysis of the peptide harboring position N53 in the heavy chain(HCDR2) of the STEAP binder revealed in all constructs a small increaseof N53 deamidation after 4 weeks in PBS, pH 7.4 at 37° C., whereas nosignificant increase at pH 6 was detected (Table 2). Oxidation of W50(LCDR2) was below 2% for all samples (Table 3). Consequently no moleculeoptimizations regarding these putative hotspots were performed.

TABLE 2 Relative quantification of the proteindegradation at position N53 (HCDR2). Deamidation (%) SampleTryptic peptide* ph 7.4, 37° C. ph 6, 40° C. No stress Molecule AGLEWVGYIS N SGSTSYNPSLK 3.4 1.3 1.2 Molecule B GLEWVGYIS N SGSTSYNPSLK3.7 1.6 1.2 Molecule C GLEWVGYIS N SGSTSYNPSLK 3.2 1.5 1.2 Molecule DGLEWVGYIS N SGSTSYNPSLK 3.3 1.5 1.4 *Position of predicted hotspot N53bold and underlined

TABLE 3 Relative quantification of the proteindegradation at position W50 (LCDR2). Tryptic Oxidation products Samplepeptide* [%] Molecule A LLIY W ASTR 1.7 Molecule B LLIY W ASTR 1.2Molecule C LLIY W ASTR 0.7 Molecule D LLIY W ASTR 1.0 *Position ofpredicted hotspot W50 bold and underlined.

Characterization of Binding Potency after Stress Using a Cell-BasedELISA

To quantify the reduction in binding potency caused by 1-4 weeks stressat either pH 7.4 or 6.0, a cell-based ELISA was employed using CHO-K1cells stably expressing human STEAP1. For this III cell-based ELISA,10,000 cells were seeded per well of a 96-well plate and incubated for18 h at 37° C., 5% CO₂. Supernatant was removed using an automatedwasher (BIOTEK), and 100 μl of a dilution series (10 μM-30 nM) ofantibody constructs in growth medium was added to each well. After 1 hof incubation at 4° C., wells were emptied and 100 μl of 0.05%glutaraldehyde in PBS added for 10 min at RT. After 4 washes withPBS/0.025% Tween20 (PBST), 100 μl of anti-human-IgG-HRP (Jackson)diluted 1:20000 in Blocking buffer (Roche) was added and platesincubated for 1 h at room temperature (RT). Wells were washed 6 timeswith PBST and signal was generated using 100 μl of TMB per well, thereaction stopped after 10 minutes with 50 μl 1 M HCl and absorbancemeasured at 450 nm. Data (Table 4) were expressed as “% binding”,dividing the binding EC50 of the stressed sample by that of theuntreated sample multiplied by 100.

Given that all tested TCB antibodies comprise two STEAP1-bindingmoieties per molecule and considering that the affinity of monovalentbinding to STEAP1 is in the range of 40-60 nM, it is conceivable thatonly molecules with two functional STEAP1-binding moieties can bedetected in this ELISA. In contrast, molecules with only one functionalSTEAP1 binding moiety are supposed to be washed away during the washingsteps described in this protocol. Therefore, the cumulative percentageof binding loss for the molecules with elevated succinimide levels ishigher than the detected protein degradation in HCDR3 (Molecules A-C).

TABLE 4 Quantification of the protein binding potency to STEAP1 after 28days at 40° C., pH 6. Relative binding potency (%) probe (after 28 daysat 40° C., pH 6) Molecule A 66 Molecule B 79 Molecule C 69 Molecule D100

Biochemical Characterization of the Vandortuzumab-Based TCB AntibodyVariants (Molecules A-D)

In order to characterize and compare their biochemical and biophysicalproperties, all TCBs with new anti-STEAP-1 antibody sequence variants(Molecules B-D) were analyzed and compared with the TCB antibodyharboring the vandortuzumab sequence (Molecule A). The results aresummarized in Table 5:

Hydrophobic Interaction Chromatography (HIC)

Apparent hydrophobicity was determined by injecting 20 μg of sample ontoa HIC-Ether-5PW (Tosoh) column equilibrated with 25 mM Na-phosphate, 1.5M ammonium sulfate, pH 7.0. Elution was performed with a linear gradientfrom 0 to 100% buffer B (25 mM Na-phosphate, pH 7.0) within 60 minutes.Retention times were compared to protein standards with knownhydrophobicity.

Thermal Stability

Samples were prepared at a concentration of 1 mg/mL in 20 mMHistidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into anoptical 384-well plate by centrifugation through a 0.4 μm filter plateand covered with paraffine oil. The hydrodynamic radius is measuredrepeatedly by dynamic light scattering on a DynaPro Plate Reader (Wyatt)while the samples are heated with a rate of 0.05° C./min from 25° C. to80° C.

FcRn Affinity Chromatography

FcRn was expressed, purified and biotinylated as described (Schlothaueret al., MAbs (2013) 5(4), 576-86). For coupling, the prepared receptorwas added to streptavidin-sepharose (GE Healthcare). The resultingFcRn-sepharose matrix was packed in a column housing. The column wasequilibrated with 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES), 140mM NaCl, pH 5.5 (eluent A) at a 0.5 ml/min flow rate. 30 μg of antibodysamples were diluted at a volume ratio of 1:1 with eluent A and appliedto the FcRn column. The column was washed with 5 column volumes ofeluent A followed by elution with a linear gradient from 20 to 100% 20mM Tris/HCl, 140 mM NaCl, pH 8.8 (eluent B) in 35 column volumes. Theanalysis was performed with a column oven at 25° C. The elution profilewas monitored by continuous measurement of the absorbance at 280 nm.Retention times were compared to protein standards with knownaffinities.

Heparin Affinity Chromatography

Heparin affinity was determined by injecting 30-50 μg of sample onto aTSKgel Heparin-5PW (Tosoh) column equilibrated with 50 mM Tris, pH 7.4.Elution was performed with a linear gradient from 0 to 100% buffer B (50mM Tris, 1M NaCl, pH 7.4 mM) within 37 minutes. Retention times werecompared to protein standards with known affinities.

No significant difference was found between any of the tested TCBs withnew anti-STEAP-1 antibody sequence variants (Molecules B-D) and themolecule harboring the vandortuzumab sequence (Molecule A) with regardto all tested biophysical and biochemical properties (Table 5). Allsamples showed only marginal aggregation and fragmentation upon stress,supporting that observed activity losses after stress exposure at pH 6(Molecules A-C) are due to chemical protein degradation at theidentified positions in the HCDR3 region.

TABLE 5 Biophysical and biochemical properties of tested variants.Thermal stability Apparent FcRn Heparin Sample (° C.) hydophobicityaffinity affinity Molecule A 58 0.10 0.61 0.85 Molecule B 58 0.16 0.690.85 Molecule C n.d. n.d. n.d. n.d. Molecule D 58 0.11 0.65 0.85 n.d.:not determined

Example 3 Functional Characterization of STEAP-1 TCB Antibody Variants

T-Cell Mediated Tumor Lysis, Induced by STEAP-1 TCB Antibody Variants

T-cell killing mediated by different STEAP-1 TCB antibody variants(Molecules A-D) was assessed on STEAP-1 expressing LnCAP cells. Humanperipheral blood mononuclear cells (PBMCs) were used as effector cellsand the killing was detected at 24 h and 48 h of incubation with thebispecific antibodies. Adherent target cells were harvested withTrypsin/EDTA, washed, and plated at a density of 30 000 cells/well usingflat-bottom 96-well plates. Cells were left to adhere overnight. PBMCswere prepared by Histopaque density centrifugation of enrichedlymphocyte preparations of heparinized blood obtained from healthy humandonors. Fresh blood was diluted with sterile PBS and layered overHistopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30minutes, room temperature), the plasma above the PBMC-containinginterphase was discarded and PBMCs transferred in a new falcon tubesubsequently filled with 50 ml of PBS. The mixture was centrifuged(400×g, 10 minutes, room temperature), the supernatant discarded and thePBMC pellet washed twice with sterile PBS (centrifugation steps 350×g,10 minutes). The resulting PBMC population was counted automatically(ViCell) and stored in RPMI1640 medium containing 10% FCS and 1%L-alanyl-L-glutamine (Biochrom. K0302) at 37° C. 5% CO₂ in cellincubator until further use (no longer than 24 h).

For the killing assay, the antibodies were added at the indicatedconcentrations (range of 0.01 μM-1 nM in triplicates. PBMCs were addedto target cells to obtain a final E:T ratio of 10:1. Target cell killingwas assessed after 24 h and 48 h of incubation at 37° C., 5% CO₂ byquantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science. #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific construct. The results after 24 h (FIG. 4A) and 48 h (FIG.4B) show that T-cell mediated tumor lysis is induced similarly by thetested molecules and that the modification of the STEAP-1 binder inMolecules B-D does not negatively affect the killing potency of themolecules.

T-Cell Activation Induced by STEAP-1 TCB Antibody Variants (Jurkat-NFATActivation Assay)

The capacity of the STEAP-1 TCB antibody variants to induce CD3-mediatedactivation of effector cells upon simultaneous binding to CD3 and humanSTEAP-1 on cells, was assessed using co-cultures of tumor antigenpositive target cells (LnCAP, 22RV1) and Jurkat-NFAT reporter cells (aCD3-expressing human acute lymphatic leukemia reporter cell line with aNFAT promoter; GloResponse Jurkat NFAT-RE-luc2P, Promega #CS176501).Upon simultaneous binding of the TCB molecule to the STEAP-1 antigen(expressed on target cells) and CD3 antigen (expressed on Jurkat-NFATreporter cells), the NFAT promoter is activated and leads to expressionof active firefly luciferase. The intensity of luminescence signal(obtained upon addition of luciferase substrate) is proportional to theintensity of CD3 activation and signaling.

For the assay, human tumor cells were harvested and viability wasdetermined using ViCell. 20 000 cells/well were plated in a flat-bottom,white-walled 96-well-plate (#655098, greiner bio-one) and dilutedantibodies or medium (for controls) was added (range of 2.6 μM-200 nM).

Subsequently, Jurkat-NFAT reporter cells were harvested and viabilityassessed using ViCell. Cells were re-suspended in cell culture mediumand added to tumor cells to obtain a final effector-to-target (E:T)ratio of 5:1 and a final volume of 100 μl per well. Cells were incubatedfor 6 h at 37° C. in a humidified incubator. At the end of theincubation time, 100 μl/well of ONE-Glo solution (Promega; 1:1 ONE-Gloand assay medium volume per well) were added to wells and incubated for10 min at room temperature in the dark. Luminescence was detected usingWALLAC Victor3 ELISA reader (PerkinElmer2030), 5 sec/well as detectiontime.

As shown in FIG. 5A-FIG. 5C, all evaluated STEAP-1 TCB antibodymolecules induce T cell cross-linking via CD3 and subsequently T cellactivation on STEAP1-expressing LnCAP (FIG. 5A) and 22Rv1 (FIG. 5B)cells. On STEAP1-negative CHO-K1 cells (FIG. 5C) no T cell activationcan be observed.

Binding of STEAP-1 TCB Antibody Variants to STEAP-1- and CD3-ExpressingCells

The binding of STEAP-1 TCB antibody variants (Molecules A-D) was tested,using STEAP-1-expressing CHO-hSTEAP1 cells (an epithelial cell linederived from hamster ovary that was transfected to stably overexpresshuman STEAP-1) and CD3-expressing Jurkat-NFAT reporter cells (Promega#CS176501).

Briefly, adherent CHO-hSTEAP1 cells were harvested, using CellDissociation Buffer (Gibco, #13151014) counted, checked for viabilityand re-suspended at 2×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA).Jurkat suspension cells were also harvested, counted and checked forviability. 100 μl of cell suspension (containing 0.2×10⁶ cells) wereincubated in round-bottom 96-well plate for 30 mn at 4° C. withincreasing concentrations of the STEAP-1 TCB antibodies (31 μM-1000 nM),washed twice with cold PBS containing 0.1% BSA (FACS buffer),re-incubated for further 30 min at 4° C. with the 1:50 pre-diluted AlexaFluor 647-conjugated AffiniPure F(ab′)₂ Fragment goat-human IgG FcγFragment Specific secondary antibody (Jackson Immuno Research Lab, AlexaFluor 647 #109-606-008, dilutions in FACS buffer) and washed twice withcold PBS 0.1% BSA.

The stained cells were re-suspended in 100 μL 2%paraformaldehyde-containing FACS Buffer and incubated for 30 min at 4°C. to fix the staining. Finally, cells were centrifuged for 4 min at350×g and 4° C., the supernatants were discarded and the cell pelletsre-suspended in 200 μl FACS Buffer. Staining was analyzed by FACS usinga FACS Canto II (Software FACS Diva). Binding curves were obtained usingGraphPadPrism6 (FIG. 6A, binding to CHO-hSTEAP1 cells; FIG. 6B, bindingto Jurkat cells).

As shown in FIG. 6A-FIG. 6B, all evaluated STEAP-1 TCB antibodymolecules show concentration-dependent binding to human CHO cellsexpressing human STEAP-1 (FIG. 6A) and to human CD3 expressed on JurkatNFAT cells (FIG. 6B), indicating that the modification of the STEAP-1binder in Molecules B-D does not negatively affect their binding toSTEAP-1 on cells.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. An antibody that binds to STEAP-1, wherein the antibody comprises aheavy chain variable region (VH) comprising a heavy chain complementarydetermining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2,and a HCDR 3 of SEQ ID NO: 4, and a light chain variable region (VL)comprising a light chain complementarity determining region (LCDR) 1 ofSEQ ID NO: 7, a LCDR 2 of SEQ ID NO: 8 and a LCDR 3 of SEQ ID NO: 9.2-37. (canceled)